Methods of synthesis and/or purification of diaminophenothiazinium compounds

ABSTRACT

Described are methods of synthesis and/or purification of certain 3,7-diamino-phenothiazin-5-ium compounds (referred to herein as “diaminophenothiazinium compounds”) including Methylthioninium Chloride (MTC) (also known as Methylene Blue), the resulting (high purity) compounds, compositions comprising such compounds (e.g., tablets, capsules), and the use of such high purity compounds. The compounds are useful in inactivating pathogens, and methods of medical treatment, prophylaxis, and diagnosis of diseases including a tauopathy, related neurological and infectious diseases.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/149,164, filed May 31, 2011; which is a divisional of U.S. patentapplication Ser. No. 12/373,216, filed Jan. 9, 2009, now U.S. Pat. No.7,956,183, issued Jun. 7, 2011; which is the U.S. National Phase ofInternational Application No. PCT/GB2007/002570, filed Jul. 10, 2007,which was published in English on Jan. 17, 2008, as WO2008/007074; andwhich claims the benefit of U.S. provisional patent application No.60/819,627, filed Jul. 11, 2006; the contents of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention pertains generally to the field of chemical synthesis andpurification, and more specifically to methods of synthesis and/orpurification of certain 3,7-diamino-phenothiazin-5-ium compounds(referred to herein as “diaminophenothiazinium compounds”) includingMethylthioninium Chloride (MTC) (also known as Methylene Blue). Thepresent invention also pertains to the resulting (high purity)compounds, compositions comprising them (e.g., tablets, capsules), andtheir use in methods of inactivating pathogens, and methods of medicaltreatment, prophylaxis, and diagnosis, etc., for example, a tauopathy; adisease of tau protein aggregation; Alzheimer's disease (AD); Pick'sdisease; Progressive Supranuclear Palsy (PSP); fronto-temporal dementia(FTD); FTD and parkinsonism linked to chromosome 17 (FTDP-17);disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration(CBD); mild cognitive impairment (MCI); skin cancer; melanoma;methemoglobinemia; a viral infection; a bacterial infection; a protozoalinfection; a parasitic infection; malaria; visceral leishmaniasis;African sleeping sickness; toxoplasmosis; giardiasis; Chagas' disease;Hepatitis C virus (HCV) infection; human immunodeficiency virus (HIV)infection; West Nile virus (WNV) infection; a synucleinopathy;Parkinson's disease (PD); dementia with Lewy bodies (DLB); multiplesystem atrophy (MSA); drug-induced parkinsonism; and pure autonomicfailure (PAF).

2. Background

Throughout this specification, including any claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps, butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and any appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

Methylthioninium Chloride (MTC) (Also Known as Methylene Blue)

Methylthioninium Chloride (MTC) (also known as Methylene blue (MB);methylthionine chloride; tetramethylthionine chloride;3,7-bis(dimethylamino) phenothiazin-5-ium chloride; C.I. Basic Blue 9;tetramethylthionine chloride; 3,7-bis(dimethylamino) phenazathioniumchloride; Swiss blue; C.I. 52015; C.I. Solvent Blue 8; aniline violet;and Urolene Blue®) is a low molecular weight (319.86), water soluble,tricyclic organic compound of the following formula:

Methylthioninium Chloride (MTC) (also known as Methylene Blue), perhapsthe most well known phenothiazine dye and redox indicator, has also beenused as an optical probe of biophysical systems, as an intercalator innanoporous materials, as a redox mediator, and in photoelectrochromicimaging.

See, for example, Colour Index (Vol. 4, 3rd edition, 1971) and Lillie etal., 1979, and references cited therein.

MTC may conveniently be considered to be an “oxidized form” whenconsidered in respect of the corresponding 10H-phenothiazine compound,N,N,N′,N′-tetramethyl-10H-phenothiazine-3,7-diamine, which mayconveniently be considered to be a “reduced form” (also known as the“leuco” form).

Synthesis and Purification

MTC was first described in a German Patent in 1877 (Badische Anilin- andSoda-Fabrik, 1877). In that patent, MTC was synthesized by nitrosylationof dimethylaniline, subsequent reduction to formN,N-dimethyl-1,4-diaminobenzene, and subsequent oxidative coupling inthe presence of hydrogen sulphide (H₂S) and iron(III) chloride (FeCl₃).

Bernthsen described subsequent studies of MTC and methods for itssynthesis (see Bernthsen, 1885a, 1885b, 1889).

Fierz-David and Blangley, 1949, also describes methods for the synthesisof MTC from dimethylaniline, as illustrated in the following scheme.

In step (a), nitrosodimethylaniline is prepared from dimethylaniline bytreatment with nitrite (NaNO₂) in aqueous acid (HCl) solution. In step(b), the nitroso compound is reduced to form p-aminodimethylaniline inaqueous acid (HCl) solution using zinc dust solution. In steps (c), (d),and (e), the p-aminodimethylaniline is oxidized in aqueous acid solutionwith another molecule of dimethylaniline, and simultaneously athiosulfonic acid group is introduced; the ring is then closed usingmanganese dioxide or copper sulfate. More specifically, a clear neutralsolution of p-aminodimethylaniline is acidified (H₂SO₄), and anon-reducing zinc chloride solution is added (ZnCl₂ with Na₂Cr₂O₇).Aluminium thiosulfate (Al₂(S₂O₃)₃) and sodium thiosulfate (Na₂S₂O₃) areadded. Sodium dichromate (Na₂Cr₂O₇) is added. The mixture is heated andaerated. Dimethylaniline is added. Sodium dichromate (Na₂Cr₂O₇) isadded. The mixture is heated, and becomes dark greenish-blue in colourdue to the formation of the thiosulfonic acid of Bindschedler green.Manganese dioxide or copper sulfate is added, and the mixture heated,and the dye precipitates from the concentrated zinc chloride solution.

Very similar synthesis methods are described in the Colour Index (Vol.4, 3rd edition, 1971), p. 4470.

Masuya et al., 1992, describe certain phenothiazine derivatives, andmethods for their preparation and use in photodynamic therapy of cancerand in immunoassays utilizing chemiluminescence. The compounds areprepared by routes similar to those discussed above.

Leventis et al., 1997, describe methods for the synthesis of certainmethylthioninium bromide (MTB) analogs, which employ phenothiazine as astarting material and which add the desired 3,7-substituents byhalogenation followed by amination. The authors assert that MTC issynthesized commercially by oxidation of N,N-dimethyl-p-phenylenediamine with Na₂Cr₂O₇ in the presence of Na₂S₂O₃, followed by furtheroxidation in the presence of N,N-dimethylaniline.

Marshall and Lewis, 1975a, describes the purification of commercial MTCand Azure B by solvent extraction and crystallisation. They assert thataqueous MTC/Azure B mixtures at a buffered pH of 9.5 can be separated byextraction with carbon tetrachloride. The carbon tetrachloride removesthe Azure B while leaving the MTC in the aqueous layer. They furtherassert that low temperature crystallisation of MTC at a concentration of0.25 N with hydrochloric acid removes metal contaminants. However, theorganic purity analysis reported therein is based on thin-layerchromatography, which is not suitable for quantification. Also, themicroanalysis for sulphated ash does not indicate a metal free sample.(The preferred technique in 1975 was atomic absorption.)

Marshall and Lewis, 1975b, describes the analysis of metal contaminantsin commercial thiazine dyes by atomic absorption spectrophotometry. Theyreport 38 samples with metal concentrations that vary widely between0.02% and 25.35% of individual samples; the metals examined were iron,potassium, sodium and zinc. They also report that other metals may bepresent which were not analysed. Aluminium, chromium, manganese, andcopper, are all involved in synthetic procedures for MTC and are almostcertain to be present. Importantly, they report large variations in themetal content of commercial samples of MTC.

Lohr et al., 1975, describes the purification of Azure B by columnchromatography, specifically by separation to isolate the desiredproduct followed by ion exchange back to the chloride. They assert thatother cationic dyes such as MTC can be purified by this method. However,column chromatography is not a suitable method for the purification ofMTC on a large scale.

Fierz-David et al., 1949, describes the synthesis of the zinc chloridedouble salt of MTC and the removal of zinc by chelating with sodiumcarbonate followed by filtration to generate zinc free methylene blue.However, the authors acknowledge that this technique cannot be used on alarge scale, because the yields are poor.

Cohn, 1899, describes methods for the synthesis of acetylleuco-methylene blue and ethylene blue, apparently using aceticanhydride and zinc powder.

Storey et al., 2006, describe recent methods for the synthesis andpurification of diaminophenothiazinium compounds such as MTC.

Uses

MTC is currently used to treat methemoglobinemia (a condition thatoccurs when the blood cannot deliver oxygen where it is needed in thebody). MTC is also used as a medical dye (for example, to stain certainparts of the body before or during surgery); a diagnostic (for example,as an indicator dye to detect certain compounds present in urine); amild urinary antiseptic; a stimulant to mucous surfaces; a treatment andpreventative for kidney stones; and in the diagnosis and treatment ofmelanoma.

MTC has been used to treat malaria either singly (Guttmann & Ehrlich,1891) or in combination with chloroquine (Schirmer et al. 2003;Rengelhausen et al. 2004).

Malaria in humans is caused by one of four protozoan species of thegenus Plasmodium: P. falciparum, P. vivax, P. ovale, or P. malariae. Allspecies are transmitted by the bite of an infected female Anophelesmosquito. Occasionally, transmission occurs by blood transfusion, organtransplantation, needle-sharing, or congenitally from mother to fetus.Malaria causes 300-500 million infections worldwide and approximately 1million deaths annually. Drug resistance, however is a major concern andis greatest for P. falciparum, the species that accounts for almost allmalaria-related deaths. Drugs or drug combinations that are currentlyrecommended for prophylaxis of malaria include chloroquine/proguanilhydrochloride, mefloquine, doxycycline and primaquine.

MTC (under the name Virostat® and subsequently Suvus®, from BioenvisionInc., New York) has shown potent viricidal activity in vitro.Specifically MTC is effective against viruses such as Hepatitis C, HIVand West Nile Virus in laboratory tests. West Nile virus (WNV) is apotentially serious illness affecting the central nervous system. Thelarge majority of infected people will show no visible symptoms or mildflu-like symptoms such as fever and headache. About one in 150 willdevelop severe symptoms including tremors, convulsions, muscle weakness,vision loss, numbness, paralysis or coma. Generally, WNV is spread bythe bite of an infected mosquito, but can also spread through bloodtransfusions, organ transplants, breastfeeding or during pregnancy frommother to child.

Suvus® is also currently in clinical trials for the treatment of chronicHepatitis C. Hepatitis C is a viral infection of the liver. The virus,HCV, is a major cause of acute hepatitis and chronic liver disease,including cirrhosis and liver cancer. HCV is spread primarily by directcontact with human blood. The major causes of HCV infection worldwideare use of unscreened blood transfusions, and re-use of needles andsyringes that have not been adequately sterilized. The World HealthOrganization has declared hepatitis C to be a global health problem,with approximately 3% of the world's population infected with HCV and itvaries considerably by region. The prevalence in the US is estimated at1.3% or approximately 3.5 million people. Egypt has a population ofapproximately 62 million and contains the highest prevalence ofhepatitis C in the world, estimated at over 20% of the nation'sapproximately 62 million people.

MTC, when combined with light, can prevent the replication of nucleicacid (DNA or RNA). Plasma, platelets and red blood cells do not containnuclear DNA or RNA. When MTC is introduced into the blood components, itcrosses bacterial cell walls or viral membrane then moves into theinterior of the nucleic acid structure. When activated with light, thecompounds then bind to the nucleic acid of the viral or bacterialpathogen, preventing replication of the DNA or RNA. Because MTC designedto inactivate pathogens, it has the potential to reduce the risk oftransmission of pathogens that would remain undetected by testing.

MTC and derivatives thereof (e.g., “diaminophenothiazinium compounds”)have been found to be useful in the treatment of tauopathies (such as,for example, Alzheimer's disease) (see, for example, Wischik, C. M., etal., 1996, 2002a).

Oral and parenteral formulations of MTC are commercially available inthe United States, usually under the name Urolene Blue®. However, theseformulations contain substantial amounts of metal impurities. Theseimpurities are highly undesirable, and many (e.g., including Al, Cr, Fe,Cu) exceed the safety limits set by European health agencies.

Consequently, there is a great need for higher purity (e.g.,pharmaceutical grade purity, e.g., a purity safe for human consumption,e.g., with low or reduced organic and/or metal impurity content)diaminophenothiazinium compounds, including MTC.

The inventors have developed methods for the synthesis ofdiaminophenothiazinium compounds (including MTC), that yield productswith extremely high purity and in particular, products with extremelylow levels of undesired impurities (both organic and metal impurities)that meet (and often exceed) the safety limits set by European healthagencies (e.g., the European Pharmacopoeia).

Without exaggeration, MTC prepared by the methods described herein isthe purest available worldwide.

SUMMARY OF THE INVENTION

One aspect of the present invention pertains to a method of synthesisand/or purification of a diaminophenothiazinium compound (DAPTC),including, for example, methylthioninium chloride (MTC), as describedherein.

Another aspect of the invention pertains to use of a method of synthesisand/or purification of a diaminophenothiazinium compound as describedherein, as part of a method of manufacturing a medicament, for example,a medicament for use in the treatment or prophylaxis of a diseasecondition (e.g., a disease condition as described herein).

Another aspect of the invention pertains to a diaminophenothiaziniumcompound, as defined herein and including, for example, methylthioniniumchloride (MTC), that is obtained by, or is obtainable by, a method ofsynthesis and/or purification as described herein and/or that has apurity as defined herein.

Another aspect of the invention pertains to a composition (e.g., apharmaceutical composition, e.g., a tablet, a capsule) comprising adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein.

Another aspect of the present invention pertains to adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein, for use in a methodof treatment (e.g., a method of treatment or prophylaxis, e.g., a methodof treatment or prophylaxis of a disease condition, as described herein)of the human or animal body by therapy.

Another aspect of the present invention pertains to the use of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein, in the manufactureof a medicament for use in the treatment or prophylaxis of a diseasecondition (e.g., a disease condition as described herein).

Another aspect of the present invention pertains to a method oftreatment or prophylaxis of a disease condition (e.g., a diseasecondition as described herein) in a patient, comprising administering tosaid patient a therapeutically-effective amount or aprophylactically-effective amount of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), that is obtained by, or is obtainable by, a method of synthesisand/or purification as described herein, and/or that has a purity asdefined herein.

In one embodiment, the disease condition is: a tauopathy; a disease oftau protein aggregation; Alzheimer's disease (AD); Pick's disease;Progressive Supranuclear Palsy (PSP); fronto-temporal dementia (FTD);FTD and parkinsonism linked to chromosome 17 (FTDP-17);disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;pallido-nigro-luysian degeneration (PNLD); cortico-basal degeneration(CBD); mild cognitive impairment (MCI); skin cancer; melanoma;methemoglobinemia; a viral infection; a bacterial infection; a protozoalinfection; a parasitic infection; malaria; visceral leishmaniasis;African sleeping sickness; toxoplasmosis; giardiasis; Chagas' disease;Hepatitis C virus (HCV) infection; human immunodeficiency virus (HIV)infection; West Nile virus (WNV) infection; a synucleinopathy;Parkinson's disease (PD); dementia with Lewy bodies (DLB); multiplesystem atrophy (MSA); drug-induced parkinsonism; or pure autonomicfailure (PAF).

Another aspect of the invention pertains to a diaminophenothiaziniumcompound, as defined herein and including, for example, methylthioniniumchloride (MTC), that is obtained by, or is obtainable by, a method ofsynthesis and/or purification as described herein, and/or that has apurity as defined herein, for use in a method of inactivating pathogens.

Another aspect of the present invention pertains to a method ofinactivating pathogens that employs an effective amount of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an HPLC chromatogram for the Medex™ starting material used inSynthesis 1. Peaks at the following retention times were observed:2.765, 2.907, 3.445, 4.033, 4.365, 5.259, and 6.230 minutes.

FIG. 2 is an HPLC chromatogram for high purity MTC-5° C. productobtained in Synthesis 4. Peaks at the following retention times wereobserved: 3.226, 5.415, and 6.335 minutes.

FIG. 3 is an HPLC chromatogram for the high purity MTC-5° C. crudeproduct obtained in Synthesis 5. Peaks at the following retention timeswere observed: 5.148, 5.418, and 6.318 minutes.

FIG. 4 is an HPLC chromatogram for high purity MTC-5° C.-recrystallisedproduct obtained in Synthesis 6. Peaks at the following retention timeswere observed: 5.439 and 6.346 minutes.

DETAILED DESCRIPTION

The Target Compounds

In general, the present invention pertains to methods for the synthesisand/or purification of certain 3,7-diamino-phenothiazin-5-ium compoundsof the following formula, collectively referred to herein as“diaminophenothiazinium compounds” (DAPTC):

wherein:

each of R¹ and R⁹ is independently selected from: —H; C₁₋₄alkyl;C₂₋₄alkenyl; and halogenated C₁₋₄alkyl;

each of R^(3NA) and R^(3NB) is independently selected from: C₁₋₄alkyl;C₂₋₄alkenyl; and halogenated C₁₋₄alkyl;

each of R^(7NA) and R^(7NB) is independently selected from: C₁₋₄alkyl;C₂₋₄alkenyl; and halogenated C₁₋₄alkyl; and

X is one or more anionic counter ions to achieve electrical neutrality.

The above structure is only one of many equivalent resonance structures,some of which are shown below, and all of which are intended to beencompassed by the above structure:

In one embodiment, the C₁₋₄alkyl groups are selected from: linearC₁₋₄alkyl groups, such as -Me, -Et, -nPr, -iPr, and -nBu; branchedC₃₋₄alkyl groups, such as -iPr, -iBu, -sBu, and -tBu; and cyclicC₃₋₄alkyl groups, such as -cPr and -cBu.

In one embodiment, the C₂₋₄alkenyl groups are selected from linearC₁₋₄alkenyl groups, such as —CH═CH₂ (vinyl) and —CH₂—CH═CH₂ (allyl).

In one embodiment, the halogenated C₁₋₄alkyl groups are selected from:—CF₃, —CH₂CF₃, and —CF₂CF₃.

In one embodiment, each of R¹ and R⁹ is independently —H, -Me, -Et, or—CF₃.

In one embodiment, each of R¹ and R⁹ is independently —H, -Me, or -Et.

In one embodiment, each of R¹ and R⁹ is independently —H.

In one embodiment, each of R¹ and R⁹ is independently -Me.

In one embodiment, each of R¹ and R⁹ is independently -Et.

In one embodiment, R¹ and R⁹ are the same.

In one embodiment, R¹ and R⁹ are different.

In one embodiment, each of R^(3NA) and R^(3NB) independently -Me, -Et,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me or-Et.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Me.

In one embodiment, each of R^(3NA) and R^(3NB) is independently -Et.

In one embodiment, R^(3NA) and R^(3NB) are the same.

In one embodiment, R^(3NA) and R^(3NB) are different.

In one embodiment, each of R^(7NA) and R^(7NB) independently -Me, -Et,-nPr, -nBu, —CH₂—CH═CH₂, or —CF₃.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me or-Et.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Me.

In one embodiment, each of R^(7NA) and R^(7NB) is independently -Et.

In one embodiment, R^(7NA) and R^(7NB) are the same.

In one embodiment, R^(7NA) and R^(7NB) are different.

In one embodiment, R^(3NA) and R^(3NB) and R^(7NA) and R^(7NB) are thesame.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are the same.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are independently selected from: —NMe₂, —NEt₂,—N(nPr)₂, —N(Bu)₂, —NMeEt, —NMe(nPr), and —N(CH₂CH═CH₂)₂.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are independently selected from: —NMe₂ and —NEt₂.

In one embodiment, each of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is independently —NMe₂.

In one embodiment, the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) are as defined herein, with the proviso thatneither group is —NMe₂.

In one embodiment, one or more of the carbon atoms is ¹¹C or ¹³C.

In one embodiment, one or more of the carbon atoms is ¹¹C.

In one embodiment, one or more of the carbon atoms is ¹³C.

In one embodiment, one or more of the nitrogen atoms is ¹⁵N.

In one embodiment, one or more or all of the carbon atoms of one or moreor all of the groups R^(3NA), R^(3NB), R^(7NA) and R^(7NB) is ¹³C.

In one embodiment, each of the groups —N(R^(3NA))(R^(3NB)) and—N(R^(7NA))(R^(7NB)) is —N(¹³CH₃)₂.

In one embodiment, each of R¹ and R⁹ is —H, and each of the groups—N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is —N(¹³CH₃)₂.

In one embodiment, each of R¹ and R⁹ is —H; each of the groups—N(R^(3NA))(R^(3NB)) and —N(R^(7NA))(R^(7NB)) is —N(¹³CH₃)₂; and X⁻ isCl⁻.

In one embodiment, X⁻ is independently a halogen anion (i.e., halide) ornitrate anion.

In one embodiment, X⁻ is independently a halogen anion (i.e., halide).

In one embodiment, X⁻ is independently Cl⁻, Br⁻, or I⁻, or NO₃ ⁻.

In one embodiment, X⁻ is independently Cl⁻, Br⁻, or I⁻.

In one embodiment, X⁻ is independently Cl⁻.

In one embodiment, the compound is in the form of a mixed salt.

Examples of diaminophenothiazinium compounds (DAPTC) include thefollowing:

One especially preferred diaminophenothiazinium compound (DAPTC) is MTC:

The Acylated Reagent Compound (ARC)

The methods of synthesis and/or purification of a diaminophenothiaziniumcompound (DAPTC), as defined herein and including, for example,methylthioninium chloride (MTC), proceed via an acylated reagentcompound (e.g., an acetylated reagent compound) (ARC). The acylatedreagent compound (ARC) is a compound of the following formula:

wherein:

R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), and R^(7NB) are as defined above; and

R¹⁰ is independently saturated aliphatic C₁₋₅alkyl, phenyl,p-methoxyphenyl, or p-nitrophenyl.

In one embodiment, R¹⁰ is independently saturated aliphatic C₁₋₅alkyl.

In one embodiment, R¹⁰ is independently saturated linear C₁₋₅alkyl.

In one embodiment, R¹⁰ is independently -Me, -Et, -nPr, -nBu, or -nPe.

In one embodiment, R¹⁰ is independently -Me (and the acylated reagentcompound is an acetylated reagent compound).

The acylated reagent compounds may conveniently be referred to as“3,7-diamino-10-acyl-phenothiazine compounds.”

When R¹⁰ is -Me, the acylated (i.e., acetylated) reagent compounds mayconveniently be referred to as “3,7-diamino-10-acetyl-phenothiazinecompounds.”

In an especially preferred embodiment, the acylated reagent compound(ARC) is 3,7-di(dimethylamino)-10-acetyl-phenothiazine, shown below:

Such acetylated reagent compounds are known, and may be prepared, forexample, from corresponding 3,7-diamino-phenothiazin-5-ium compounds,for example, from methylthioninium chloride (MTC), using the followingsynthesis scheme, which is also illustrated in the examples below, or byusing an analogous scheme.

Acylated (e.g., acetylated) reagent compounds may also be obtained usingthe following synthesis scheme or an analogous scheme. See, for example,Cohn, G., 1900.

Alternative reducing agents for the first step above have also beenused, including phenylhydrazine. See, for example, Drew et al., 1933.

Acylated (e.g., acetylated) reagent compounds may also be obtained usingthe following synthesis scheme or an analogous scheme. In regard to thefirst two steps of this synthesis scheme, see, for example, Tomilin etal., 1996.

Acylated (e.g., acetylated) reagent compounds may also be obtained usingthe following synthesis scheme or an analogous scheme. In regard to thefirst step of this synthesis scheme, see, for example, Leventis et al.,1997.

Acylated (e.g., acetylated) reagent compounds may also be obtained usingthe following synthesis scheme or an analogous scheme. In regard to thefirst step of this synthesis scheme, see, for example, Gilman et al.,1944.

Whatever synthesis route is taken, an acylation (e.g., acetylation) stepis involved, specifically, a step of acylating (e.g., acetylating) anupstream precursor of the acylated (e.g., acetylated) reagent compound,and, ultimately, an acylated (e.g., acetylated) reagent compound (ARC)is obtained.

The Non-Acylated Precursor of the Acylated Reagent Compound (NAPARC)

In one embodiment, the acylated reagent compound (ARC) (e.g., theacetylated precursor of the acetylated reagent compound) is obtainedfrom a corresponding non-acylated precursor of the acylated reagentcompound (NAPARC) (e.g., a corresponding non-acetylated precursor of theacetylated reagent compound).

In one embodiment, the non-acylated precursor of the acylated reagentcompound (NAPARC) (e.g., the acetylated precursor of the acetylatedreagent compound) is a compound of the following formula, wherein R¹,R⁹, R^(3NA), R^(3NB), R^(7NA), and R^(7NB) are as defined herein:

In an especially preferred embodiment, the non-acylated precursor of theacylated reagent compound (NAPARC) is3,7-di(dimethylamino)-10H-phenothiazine, shown below:

The Oxidized Precursor of the Non-Acylated Precursor of the AcylatedReagent Compound (OPNAPARC)

In one embodiment, the non-acylated precursor of the acylated reagentcompound (NAPARC) (e.g., a non-acetylated precursor of the acetylatedreagent compound) is obtained from a corresponding oxidized precursor ofthe non-acylated precursor of the acylated reagent compound (OPNAPARC)(e.g., a corresponding oxidized precursor of the non-acetylatedprecursor of the acetylated reagent compound).

In one embodiment, the oxidized precursor of the non-acylated precursorof the acylated reagent compound (OPNAPARC) (e.g., the oxidizedprecursor of the acetylated precursor of the acetylated reagentcompound) is a compound of the following formula, wherein R¹, R⁹,R^(3NA), R^(3NB), R^(7NA), and R^(7NB) are as defined herein, andwherein X^(a) is as defined for X, and may be the same as or differentthan X:

In an especially preferred embodiment, the oxidized precursor of thenon-acylated precursor of the acylated reagent compound (OPNAPARC) ismethyl thioninium chloride (MTC), shown below:

Upstream Precursors

In an alternative approach, the acylation step is performed furtherupstream, to give an acylated upstream precursor of the acylated reagentcompound (AUPARC) (e.g., an acetylated precursor of the acetylatedreagent compound) from a corresponding non-acylated upstream precursorof the acylated reagent compound (NAUPARC) (e.g., a correspondingnon-acetylated upstream precursor of the acetylated reagent compound).

The Non-Acylated Upstream Precursor of the Acylated Reagent Compound(NAUPARC)

For example, in one embodiment, the non-acylated upstream precursor ofthe acylated reagent compound (e.g., the non-acylated upstream precursorof the acetylated reagent compound) falls within one of the followingclasses of compounds, wherein R¹ and R⁹ are as defined above:

In one preferred embodiment, the non-acylated upstream precursor of theacylated reagent compound (NAUPARC) (e.g., the non-acetylated upstreamprecursor of the acetylated reagent compound) is a compound of thefollowing formula, wherein R¹ and R⁹ are as defined herein:

An example of a preferred non-acylated upstream precursor of theacylated reagent compound (NAUPARC) is 3,7-dinitro-10H-phenothiazine,shown below:

The Acylated Upstream Precursor of the Acylated Reagent Compound(AUPARC)

In one preferred embodiment, the acylated upstream precursor of theacylated reagent compound (AUPARC) (e.g., the acetylated upstreamprecursor of the acetylated reagent compound) is a compound of thefollowing formula, wherein R¹, R⁹, and R¹⁰ as defined herein:

An example of a preferred upstream acylated precursor of the acylatedreagent compound (AUPARC) is 3,7-dinitro-10-acetyl-phenothiazine, shownbelow:

Acylated Compounds Generally

Without wishing to be bound by any particular theory, the inventorsbelieve that the use of an acylation step (e.g., an acetylation step),and the formation of an acylated reagent compound (ARC) (e.g., anacetylated reagent compound) or an acylated upstream precursor of theacylated reagent compound (AUPARC) (e.g., an acetylated upstreamprecursor of the acetylated reagent compound), facilitates the easyremoval of many undesired impurities and by-products, and leads,ultimately, to an acylated reagent compound (ARC) (e.g., an acetylatedreagent compound) with higher purity, which, in turn, leads to a targetdiaminophenothiazinium compound (DAPTC) with a higher purity.

For example, Azure B is an undesired impurity often found in samples ofmethylthioninium chloride (MTC). Commercially available Medex™ containsmore than 5% by weight of Azure B (see analysis details below). Removalof Azure B from mixtures of MTC and Azure B is particularly difficult.However, when such a mixture is used as a starting material, and anacetylation step is employed, acetylation of the Azure B leads to adi-acetylated water-soluble by-product that may easily be separated fromthe desired organic-soluble acetylated reagent compound, for example, bywashing with water and recrystallisation. For example, the synthesis andprecipitation of the desired acetylated reagent compound, optionallyfollowed by recrystallisation of the precipitated desired acetylatedreagent compound, readily achieves removal of much or most or all of theundesired Azure B (presumably in the form of the di-acetylated Azure Bby-product) and other organic impurities from the desired acetylatedreagent compound.

Azure A and Azure C, among other impurities, are similarly reduced bythe same mechanism.

Methods Generally

For convenience, many of methods described herein may be illustratedusing a preferred example as represented in the following scheme.

Synthesis and/or Purification of Diaminophenothiazinium Compounds—A

Thus, one aspect of the present invention pertains to a method for thesynthesis and/or purification of a diaminophenothiazinium compound(DAPTC), as defined herein and including, for example, methylthioniniumchloride (MTC), which method comprises at least the following steps, inorder:

purifying (PUR) a corresponding acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound; and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

purifying (PUR) a corresponding acetylated reagent compound;

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound; and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound.

In one embodiment, the method is as illustrated by the following scheme,in which R¹, R⁹, R¹⁰, R^(3NA), R^(3NB), R^(7NA), R^(7NB), and X are asdefined herein.

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound (DAPTC), as definedherein and including, for example, methylthioninium chloride (MTC),which method comprises at least the following steps, in order:

purifying (PUR) a corresponding acylated reagent compound (ARC) of thefollowing formula:

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound of the following formula:

and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound(DAPTC), as defined herein and including, for example, methylthioniniumchloride (MTC), which method comprises at least the following steps, inorder:

purifying (PUR) a corresponding acetylated reagent compound of thefollowing formula:

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound of the following formula:

and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For the avoidance of doubt, the word “corresponding” in the phrases“corresponding acylated reagent compound” and “corresponding decylatedcompound” is intended to mean “corresponding to the targetdiaminophenothiazinium compound,” and so the groups R¹, R⁹, R^(3NA),R^(3NB), R^(7NA), R^(7NB) of the acylated reagent compound and thedecylated compound, if present, are the same as the corresponding groupsR¹, R⁹, R^(3NA), R^(3NB), R^(7NA), R^(7NB) of the targetdiaminophenothiazinium compound.

The acylated (e.g., acetylated) reagent compound used in said purifying(PUR) step may be obtained from any source or may be obtained using anymethod of synthesis, for example, using a method of synthesis asdescribed herein.

In a preferred embodiment, the method is a method for the synthesisand/or purification of methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

purifying (PUR) 3,7-di(dimethylamino)-10-acetyl-phenothiazine;

deacylating (DAC) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine togive 3,7-di(dimethylamino)-10H-phenothiazine; and

oxidizing (OX) said 3,7-di(dimethylamino)-10H-phenothiazine to give saidmethylthioninium chloride (MTC).

An example of this embodiment is illustrated in the following scheme.

Synthesis and/or Purification of Diaminophenothiazinium Compounds—A+B

In one embodiment, the acylated reagent compound (ARC) used in saidpurifying (PUR) step is obtained by acylating the non-acylated (e.g.,N¹⁰-unsubstituted) precursor of the corresponding acylated reagentcompound (NAPARC).

For example, in one embodiment, where R¹⁰ is -Me, the acetylated reagentcompound used in said purifying (PUR) step is obtained by acetylatingthe non-acetylated (e.g., N¹⁰-unsubstituted) precursor of thecorresponding acetylated reagent compound.

Thus, in one embodiment, the method further comprises the followingstep, before said purifying (PUR) step:

acylating (AC1) a corresponding non-acylated precursor of said acylatedreagent compound (NAPARC) to give said acylated reagent compound (ARC).

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said purifying (PUR) step:

acylating (AC1) a corresponding non-acetylated precursor of saidacetylated reagent compound to give said acetylated reagent compound.

In one embodiment, the method further comprises the following step,before said purifying (PUR) step:

acylating (AC1) a corresponding non-acylated precursor of an acylatedreagent compound (NAPARC) to give said acylated reagent compound (ARC),

wherein said non-acylated precursor (NAPARC) is a compound of thefollowing formula:

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said purifying (PUR) step:

acylating (AC1) a corresponding non-acetylated precursor of saidacetylated reagent compound to give said acetylated reagent compound,

wherein said non-acetylated precursor is a compound of the followingformula:

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound (DAPTC), as definedherein and including, for example, methylthioninium chloride (MTC),which method comprises at least the following steps, in order:

acylating (AC1) a corresponding non-acylated precursor of acorresponding acylated reagent compound (NAPARC) to give a correspondingacylated reagent compound (ARC);

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound; and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

acylating (AC1) a corresponding non-acetylated precursor of acorresponding acetylated reagent compound to give said acetylatedreagent compound;

purifying (PUR) said acetylated reagent compound;

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound; and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

In one embodiment, the method is as illustrated by the following scheme,in which R¹, R⁹, R¹⁰, R^(3NA), R^(3NB), R^(7NA), R^(7NB), and X are asdefined herein.

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound, as defined herein andincluding, for example, methylthioninium chloride (MTC), which comprisesat least the following steps, in order:

acylating (AC1) a corresponding non-acylated precursor of acorresponding acylated reagent compound (NAPARC) to give saidcorresponding acylated reagent compound (ARC), wherein said non-acylatedprecursor (NAPARC) is a compound of the following formula:

and wherein said acylated reagent compound (ARC) is a compound of thefollowing formula:

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said purified acylated reagent compound (ARC) to givea corresponding deacylated compound of the following formula:

and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), which comprises at least the following steps, in order:

acylating (AC1) a corresponding non-acetylated precursor of acorresponding acetylated reagent compound to give said acetylatedreagent compound;

wherein said non-acetylated precursor is a compound of the followingformula:

wherein said acetylated reagent compound is a compound of the followingformula:

purifying (PUR) said acetylated reagent compound;

deacylating (DAC) said purified acetylated reagent compound to give acorresponding deacetylated compound of the following formula:

and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For the avoidance of doubt, the word “corresponding” in the phrases“corresponding non-acylated precursor of a corresponding acylatedreagent compound,” “corresponding acylated reagent compound” and“corresponding decylated compound” is intended to mean “corresponding tothe target diaminophenothiazinium compound,” and so the groups R¹, R⁹,R^(3NA), R^(3NB), R^(7NA), R^(7NB) of the non-acylated precursor, theacylated reagent compound, and the decylated compound, if present, arethe same as the corresponding groups R¹, R⁹, R^(3NA), R^(3NB), R^(7NA),R^(7NB) of the target diaminophenothiazinium compound.

The non-acylated (e.g., non-acetylated) (e.g., N¹⁰-unsubstituted)precursor of an acylated (e.g., acetylated) reagent compound used insaid acylating (AC1) step may be obtained from any source or may beobtained using any method of synthesis, for example, using a method asdescribed herein.

In a preferred embodiment, the method is a method for the synthesisand/or purification of methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

acylating (AC1) 3,7-di(dimethylamino)-10H-phenothiazine to give3,7-di(dimethylamino)-10-acetyl-phenothiazine;

purifying (PUR) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine;

deacylating (DAC) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine togive 3,7-di(dimethylamino)-10H-phenothiazine; and

oxidizing (OX) said 3,7-di(dimethylamino)-10H-phenothiazine to give saidmethylthioninium chloride (MTC).

An example of this embodiment is illustrated in the following scheme.

Synthesis and/or Purification of Diaminophenothiazinium Compounds—A+B+C

In one embodiment, the acylated (e.g., acetylated) reagent compound usedin said acylating (AC1) step is obtained by reducing a correspondingdiaminophenothiazinium compound.

Thus, in one embodiment, the method further comprises the followingstep, before said acylating (AC1) step:

reducing (RED) a corresponding oxidized precursor of said non-acylatedprecursor of said acylated reagent compound (OPNAPARC) to give saidnon-acylated precursor of said acylated reagent compound.

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said acylating (AC1) step:

reducing (RED) a corresponding oxidized precursor of said non-acetylatedprecursor of said acetylated reagent compound to give saidnon-acetylated precursor of said acetylated reagent compound.

In one embodiment, the method further comprises the following step,before said acylating (AC1) step:

reducing (RED) a corresponding oxidized precursor of said non-acylatedprecursor of said acylated reagent compound (OPNAPARC) to give saidnon-acylated precursor of said acylated reagent compound (NAPARC),

wherein said oxidized precursor (OPNARC) is a compound of the followingformula, wherein X^(a) is as defined for X, and may be the same as ordifferent than X:

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said acylating (AC1) step:

reducing (RED) a corresponding oxidized precursor of said non-acetylatedprecursor of said acetylated reagent compound to give saidnon-acetylated precursor of said acetylated reagent compound,

wherein said oxidized precursor is a compound of the following formula,wherein X^(a) is as defined for X, and may be the same as or differentthan X:

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound, as defined herein andincluding, for example, methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

reducing (RED) a corresponding oxidized precursor of a correspondingnon-acylated precursor of a corresponding acylated reagent compound(OPNAPARC) to give said corresponding non-acylated precursor of saidacylated reagent compound (NAPARC);

acylating (AC1) said non-acylated precursor (NAPARC) to give saidacylated reagent compound (ARC);

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound; and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

reducing (RED) a corresponding oxidized precursor of a correspondingnon-acetylated precursor of a corresponding acetylated reagent compoundto give said non-acetylated precursor of said acetylated reagentcompound;

acylating (AC1) said non-acetylated precursor of said acetylated reagentcompound to give said acetylated reagent compound;

purifying (PUR) said acetylated reagent compound;

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound; and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

In one embodiment, the method is as illustrated by the following scheme,in which R¹, R⁹, R¹⁰, R^(3NA), R^(3NB), R^(7NA), R^(7NB), and X are asdefined herein, and X^(a) is as defined for X, and may be the same as ordifferent than X. In one embodiment, X^(a) and X are the same.

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound, as defined herein andincluding, for example, methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

reducing (RED) a corresponding oxidized precursor of a correspondingnon-acylated precursor of a corresponding acylated reagent compound(OPNAPARC) to give said non-acylated precursor of said acylated reagentcompound (NAPARC), wherein said oxidized precursor (OPNAPARC) is acompound of the following formula:

and wherein said non-acylated precursor (NAPARC) is a compound of thefollowing formula:

acylating (AC1) said non-acylated precursor of said acylated reagentcompound (NAPARC) to give said acylated reagent compound (ARC), whereinsaid acylated reagent compound (ARC) is a compound of the followingformula:

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound of the following formula:

and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method comprises at leastthe following steps, in order:

reducing (RED) a corresponding oxidized precursor of a correspondingnon-acetylated precursor of a corresponding acetylated reagent compoundto give said non-acetylated precursor of said acetylated reagentcompound;

wherein said oxidized precursor is a compound of the following formula:

wherein said non-acetylated precursor is a compound of the followingformula:

acylating (AC1) said non-acetylated precursor of said acetylated reagentcompound to give said acetylated reagent compound, wherein saidacetylated reagent compound is a compound of the following formula:

purifying (PUR) said acetylated reagent compound;

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound of the following formula:

and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

Corresponding oxidized precursor of a corresponding non-acetylatedprecursor of a corresponding acetylated reagent compound.

For the avoidance of doubt, the word “corresponding” in the phrases “acorresponding oxidized precursor of a corresponding non-acetylatedprecursor of a corresponding acetylated reagent compound,”“corresponding non-acylated precursor of a corresponding acylatedreagent compound,” “corresponding acylated reagent compound” and“corresponding decylated compound” is intended to mean “corresponding tothe target diaminophenothiazinium compound,” and so the groups R¹, R⁹,R^(3NA), R^(3NB), R^(7NA), R^(7NB) of the oxidized precursor, thenon-acylated precursor, the acylated reagent compound, and the decylatedcompound, if present, are the same as the corresponding groups R¹, R⁹,R^(3NA), R^(3NB), R^(7NA), R^(7NB) of the target diaminophenothiaziniumcompound.

The oxidized precursor of the non-acylated precursor of the acylatedreagent compound (OPNAPARC) (e.g., the oxidized precursor of thenon-acetylated precursor of an acetylated reagent compound) used in saidreducing (RED) step may be obtained from any source or may be obtainedusing any method of synthesis, for example, using a method as describedherein.

In a preferred embodiment, the method is a method for the synthesisand/or purification of methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

reducing (RED) methylthioninium chloride (MTC) to give3,7-di(dimethylamino)-10H-phenothiazine;

acylating (AC1) said 3,7-di(dimethylamino)-10H-phenothiazine to give3,7-di(dimethylamino)-10-acetyl-phenothiazine;

purifying (PUR) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine;

deacylating (DAC) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine togive 3,7-di(dimethylamino)-10H-phenothiazine; and

oxidizing (OX) said 3,7-di(dimethylamino)-10H-phenothiazine to give saidmethylthioninium chloride (MTC).

An example of this embodiment is illustrated in the following scheme.

In one embodiment, the oxidized precursor of the non-acetylatedprecursor of an acetylated reagent compound (e.g., methylthioniniumchloride (MTC)) is provided in an impure form, for example, as a mixturecomprising MTC and one or more organic impurities, for example, morethan 5% (or more than 4%; or more than 3%) of one or more organicimpurities, for example, one or more of Azure B, Azure A, Azure C, andMVB, for example, more than 5% (or more than 4%; or more than 3%) of oneor more of Azure B, Azure A, Azure C, and MVB.

For example, in one embodiment, the method is as illustrated by thefollowing scheme, in which R¹, R⁹, R¹⁰, R^(3NA), R^(3NB), R^(7NA),R^(7NB), and X are as defined herein, and X^(a) is as defined for X, andmay be the same as or different than X. In one embodiment, X^(a) and Xare the same.

Synthesis and/or Purification of Diaminophenothiazinium Compounds—A+D

In one embodiment, the acylated reagent compound (ARC) used in saidpurifying (PUR) step is obtained by converting (CON) an acylatedupstream precursor of the acylated reagent compound (AUPARC) to theacylated reagent compound (ARC).

For example, in one embodiment, where R¹⁰ is -Me, the acetylated reagentcompound used in said purifying (PUR) step is obtained by converting(CON) an acetylated upstream precursor of the corresponding acetylatedreagent compound to the acetylated reagent compound.

Thus, in one embodiment, the method further comprises the followingstep, before said purifying (PUR) step:

converting (CON) a corresponding acylated upstream precursor of saidacylated reagent compound (AUPARC) to said corresponding acylatedreagent compound (ARC).

For example, in one embodiment, the method further comprises thefollowing step, before said purifying (PUR) step:

converting (CON) a corresponding acetylated upstream precursor of saidacetylated reagent compound to said corresponding acetylated reagentcompound.

In one embodiment, the method further comprises the following step,before said purifying (PUR) step:

converting (CON) a corresponding acylated upstream precursor of saidacylated reagent compound (AUPARC) to said corresponding acylatedreagent compound (ARC),

wherein said acylated upstream precursor of said acylated reagentcompound (AUPARC) is a compound of the following formula:

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said purifying (PUR) step:

converting (CON) a corresponding acetylated upstream precursor of saidacetylated reagent compound to said corresponding acetylated reagentcompound,

wherein said acetylated upstream precursor of said acetylated reagentcompound is a compound of the following formula:

Thus, in one embodiment (“acetyl”), the method is a method for thesynthesis and/or purification of a diaminophenothiazinium compound, asdefined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

converting (CON) a corresponding acylated upstream precursor of acorresponding acylated reagent compound (AUPARC) to said acylatedreagent compound (ARC);

purifying (PUR) said acylated reagent compound (ARC),

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound; and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

converting (CON) a corresponding acetylated upstream precursor of acorresponding acetylated reagent compound to said acetylated reagentcompound;

purifying (PUR) said acetylated reagent compound,

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound; and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

In one embodiment, the method is as illustrated by the following scheme,in which R¹, R⁹, R¹⁰, R^(3NA), R^(3NB), R^(7NA), R^(7NB), and X are asdefined herein.

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound, as defined herein andincluding, for example, methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

converting (CON) a corresponding acylated upstream precursor of acorresponding acylated reagent compound (AUPARC) to said acylatedreagent compound (ARC),

wherein said acylated upstream precursor (AUPARC) is a compound of thefollowing formula:

and wherein said acylated reagent compound (ARC) is a compound of thefollowing formula:

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound of the following formula:

and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For example, in one embodiment (“acetyl”), the method is a method forthe synthesis and/or purification of a diaminophenothiazinium compound,as defined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

converting (CON) a corresponding acetylated upstream precursor of acorresponding acetylated reagent compound to said acetylated reagentcompound,

wherein said acetylated upstream precursor of a corresponding acetylatedreagent compound (AUPARC) is a compound of the following formula:

wherein said acetylated reagent compound is a compound of the followingformula:

purifying (PUR) said acetylated reagent compound;

deacylating (DAC) said acetylated reagent compound to give acorresponding deacetylated compound of the following formula:

and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For the avoidance of doubt, the word “corresponding” in the phrases “acorresponding acylated upstream precursor of a corresponding acylatedreagent compound,” “a corresponding acylated reagent compound,” and “acorresponding deacylated compound” is intended to mean “corresponding tothe target diaminophenothiazinium compound,” and so the groups R¹, R⁹,R^(3NA), R^(3NB), R^(7NA), and R^(7NB) of the acylated upstreamprecursor, the acylated reagent compound, and the deacylated compound,if present, are the same as the corresponding groups R¹, R⁹, R^(3NA),R^(3NB), R^(7NA), R^(7NB), of the target diaminophenothiaziniumcompound.

In a preferred embodiment, the method is a method for the synthesisand/or purification of methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

converting (CON) 3,7-dinitro-10-acetyl-phenothiazine to3,7-di(dimethylamino)-10-acetyl-phenothiazine;

purifying (PUR) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine;

deacylating (DAC) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine togive 3,7-di(dimethylamino)-10H-phenothiazine; and

oxidizing (OX) said 3,7-di(dimethylamino)-10H-phenothiazine to give saidmethylthioninium chloride (MTC).

An example of this embodiment is illustrated in the following scheme.

Synthesis and/or Purification of Diaminophenothiazinium Compounds—A+D+E

In one embodiment, the acylated upstream precursor of a correspondingacylated reagent compound (AUPARC) used in said purifying (PUR) step isobtained by acylating the non-acylated (e.g., N¹⁰-unsubstituted)upstream precursor of the corresponding acylated reagent compound(NAUPARC).

For example, in one embodiment, where R¹⁰ is -Me, the acetylatedupstream precursor of a corresponding acetylated reagent compound usedin said purifying (PUR) step is obtained by acetylating thenon-acetylated (e.g., N¹⁰-unsubstituted) upstream precursor of thecorresponding acetylated reagent compound.

Thus, in one embodiment, the method further comprises the followingstep, before said purifying (PUR) step:

acylating (AC2) a corresponding non-acylated upstream precursor of thecorresponding acylated reagent compound (NAUPARC) to give said acylatedupstream precursor of said corresponding acylated reagent compound(AUPARC).

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said purifying (PUR) step:

acylating (AC2) a corresponding non-acetylated upstream precursor of thecorresponding acetylated reagent compound to give said acetylatedupstream precursor of said corresponding acetylated reagent compound.

In one embodiment, the method further comprises the following step,before said purifying (PUR) step:

acylating (AC2) a corresponding non-acylated upstream precursor of acorresponding acylated reagent compound (NAUPARC) to give said acylatedupstream precursor (AUPARC),

wherein said non-acylated upstream precursor (NAUPARC) is a compound ofthe formula:

For example, in one embodiment (“acetyl”), the method further comprisesthe following step, before said purifying (PUR) step:

acylating (AC2) a corresponding non-acetylated upstream precursor of acorresponding acetylated reagent compound to give said acetylatedupstream precursor of said corresponding acetylated reagent compound,

wherein said non-acetylated upstream precursor is a compound of theformula:

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound (DAPTC), as definedherein and including, for example, methylthioninium chloride (MTC),which method comprises at least the following steps, in order:

acylating (AC2) a corresponding non-acylated upstream precursor of acorresponding acylated reagent compound (NAUPARC) to give said acylatedupstream precursor of said corresponding acylated reagent compound(AUPARC);

converting (CON) said acylated upstream precursor (AUPARC) to saidcorresponding acylated reagent compound (ARC);

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound; and

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

In one embodiment (“acetyl”), the method is a method for the synthesisand/or purification of a diaminophenothiazinium compound (DAPTC), asdefined herein and including, for example, methylthioninium chloride(MTC), which method comprises at least the following steps, in order:

acylating (AC2) a corresponding non-acetylated upstream precursor of acorresponding acetylated reagent compound (NAUPARC) to give saidacetylated upstream precursor of said corresponding acetylated reagentcompound (AUPARC);

converting (CON) said acetylated upstream precursor (AUPARC) to saidcorresponding acetylated reagent compound (ARC);

purifying (PUR) said acetylated reagent compound (ARC);

deacylating (DAC) said acetylated reagent compound (ARC) to give acorresponding deacetylated compound; and

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

In one embodiment, the method is as illustrated by the following scheme,in which R¹, R⁹, R¹⁰, R^(3NA), R^(3NB), R^(7NA), R^(7NB), and X are asdefined herein.

Thus, in one embodiment, the method is a method for the synthesis and/orpurification of a diaminophenothiazinium compound (DAPTC), as definedherein and including, for example, methylthioninium chloride (MTC),which method comprises at least the following steps, in order:

acylating (AC2) a corresponding non-acylated upstream precursor of acorresponding acylated reagent compound (NAUPARC) to give said acylatedupstream precursor (AUPARC), wherein said non-acylated upstreamprecursor (NAUPARC) is a compound of the formula:

and wherein said acylated upstream precursor (AUPARC) is a compound ofthe following formula:

converting (CON) said acylated upstream precursor (AUPARC) to saidacylated reagent compound (ARC), wherein said acylated reagent compound(ARC) is a compound of the following formula:

purifying (PUR) said acylated reagent compound (ARC);

deacylating (DAC) said acylated reagent compound (ARC) to give acorresponding deacylated compound of the following formula:

oxidizing (OX) said deacylated compound to give saiddiaminophenothiazinium compound (DAPTC).

Thus, in one embodiment (“acetyl”), the method is a method for thesynthesis and/or purification of a diaminophenothiazinium compound(DAPTC), as defined herein and including, for example, methylthioniniumchloride (MTC), which method comprises at least the following steps, inorder:

acylating (AC2) a corresponding non-acetylated upstream precursor of acorresponding acetylated reagent compound to give said acetylatedupstream precursor of said corresponding acetylated reagent compound,

wherein said non-acetylated upstream precursor is a compound of theformula:

and wherein said acetylated upstream precursor (AUPARC) is a compound ofthe following formula:

converting (CON) said acetylated upstream precursor (AUPARC) to saidacetylated reagent compound (ARC),

wherein said acetylated upstream precursor (AUPARC) is a compound of thefollowing formula:

purifying (PUR) said acetylated reagent compound;

deacylating (DAC) said acetylated reagent compound (ARC) to give acorresponding deacetylated compound of the following formula:

oxidizing (OX) said deacetylated compound to give saiddiaminophenothiazinium compound (DAPTC).

For the avoidance of doubt, the word “corresponding” in the phrases “acorresponding non-acetylated upstream precursor of a correspondingacetylated reagent compound,” “a corresponding acylated upstreamprecursor of a corresponding acylated reagent compound,” “acorresponding acylated reagent compound,” and “a correspondingdeacylated compound” is intended to mean “corresponding to the targetdiaminophenothiazinium compound,” and so the groups R¹, R⁹, R^(3NA),R^(3NB), R^(7NA), and R^(7NB) of the non-acylated upstream precursor,the acylated upstream precursor, the acylated reagent compound, and thedeacylated compound, if present, are the same as the correspondinggroups R¹, R⁹, R^(3NA), R^(3NB), R^(7NA), R^(7NB) of the targetdiaminophenothiazinium compound.

The non-acylated (e.g., acetylated) upstream precursor of the acylated(e.g., acetylated) reagent compound used in said converting (CON) stepmay be obtained from any source or may be obtained using any method ofsynthesis, for example, using a method of synthesis as described herein.

In a preferred embodiment, the method is a method for the synthesisand/or purification of methylthioninium chloride (MTC), which methodcomprises at least the following steps, in order:

acylating (AC2) 3,7-dinitro-10H-phenothiazine to give3,7-dinitro-10-acetyl-phenothiazine;

converting (CON) said 3,7-dinitro-10-acetyl-phenothiazine to3,7-di(dimethylamino)-10-acetyl-phenothiazine;

purifying (PUR) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine;

deacylating (DAC) said 3,7-di(dimethylamino)-10-acetyl-phenothiazine togive 3,7-di(dimethylamino)-10H-phenothiazine; and

oxidizing (OX) said 3,7-di(dimethylamino)-10H-phenothiazine to give saidmethylthioninium chloride (MTC).

An example of this embodiment is illustrated in the following scheme.

Optional Additional Purification Steps

Optionally, after said reducing (RED) step, and before said acylating(AC1) step, said non-acylated precursor obtained in said reducing (RED)step is purified (PUR^(RED-AC1)). This is preferred.

Alternatively, said reducing (RED) step and said acylating (AC1) stepare performed in sequence and without isolation or purification of saidnon-acylated precursor obtained in said reducing (RED) step. In thisway, both steps may be performed in a “one-pot” procedure.

Optionally, after said deacylating (DAC) step, and before said oxidizing(OX) step, said deacylated compound obtained in said deacylating (DAC)step is purified (PUR^(DAC-OX)). However, this is not preferred.

Alternatively, said deacylating (DAC) step and said oxidizing (OX) stepare performed in sequence and without isolation or purification of saiddeacylated compound obtained in said deacylating (DAC) step. In thisway, both steps may be performed in a “one-pot” procedure. This ispreferred.

Optionally, after said oxidizing (OX) step, said diaminophenothiaziniumcompound obtained in said oxidizing (OX) step is purified (PUR^(OX)).This purification step is discussed in more detail below.

Optionally, after said acylating (AC2) step, and before said converting(CON) step, said acylated upstream precursor obtained in said acylating(AC2) is purified (PUR^(AC2-CON)).

The Reducing (RED) Step

The reducing (RED) step may be performed using any suitable reducingreagents and/or conditions.

In one embodiment, the reducing (RED) step is by reaction with one ormore reducing reagents, under reducing step conditions.

In one embodiment, the one or more reducing reagents include sodiumborohydride (NaBH₄).

In one embodiment, the one or more reducing reagents includemethylhydrazine (MeNHNH₂).

In one embodiment, the one or more reducing reagents include hydrazine(NH₂NH₂) and/or hydrazine hydrate (NH₂NH₂.H₂O).

In one embodiment, the reducing step conditions include a reactiontemperature for a reaction time.

In one embodiment, the reaction temperature is about 10-70° C.

In one embodiment, the reaction temperature is about 30-50° C.

In one embodiment, the reaction temperature is about 40° C.

In one embodiment, the reaction time is about 10 minutes to 6 hours.

In one embodiment, the reaction time is about 10 minutes to 1 hour.

In one embodiment, the reaction time is about 20 minutes to 3 hours.

In one embodiment, the reaction time is about 20 minutes to 40 minutes.

In one embodiment, the reaction time is about 1 hour.

In one embodiment, the reaction time is about 30 minutes.

In one embodiment, the reducing step conditions include the use of areducing step solvent.

In one embodiment, the reducing step solvent is ethanol.

In one embodiment, the reducing step solvent is acetonitrile.

In one embodiment, the reducing step conditions include the use of aninert atmosphere.

In one embodiment, the inert atmosphere is argon (e.g., dry argon).

In one embodiment, the inert atmosphere is nitrogen (e.g., drynitrogen).

For example, in one embodiment, the diaminophenothiazinium compound (˜27mmol), ethanol (75 cm³) and sodium borohydride (˜53 mmol) are combinedunder an atmosphere of argon, and the resulting mixture is heated at˜40° C. for ˜1 hour with stirring. The resulting suspension is thencooled to ˜5° C. and filtered under argon, washed with ethanol (˜20cm³), and dried under vacuum to give desired reduced product.

For example, in one embodiment, the diaminophenothiazinium compound (˜27mmol), acetonitrile (50 cm³) and sodium borohydride (˜35 mmol) arecombined under an atmosphere of argon, and the resulting mixture isheated at ˜65° C. for ˜20 minutes with stirring. The resultingsuspension is then cooled to ˜5° C. to give desired reduced product.

In another example, methylhydrazine (˜59 mmol; ˜54 mmol) is used insteadof sodium borohydride.

In another example, hydrazine monohydrate (˜120 mmol; ˜59 mmol) is usedinstead of sodium borohydride.

The Acylating (AC1) Step and the Acylating (AC2) Step

The acylating (AC1) step and the acylating (AC2) step may be performedusing any suitable acylating (e.g., acetylating) reagents and/orconditions.

In one embodiment, the acylating (AC1) step and the acylating (AC2) stepis by reaction with one or more acylating reagents, under acylating stepconditions.

In one embodiment, the acylating (AC1) step is an acetylating step.

In one embodiment, the acylating (AC2) step is an acetylating step.

In one embodiment, the one or more acylating reagents include aceticanhydride (CH₃CO)₂O.

In one embodiment, the acylating step conditions include the use of anacylating step solvent.

In one embodiment, the acylating step solvent is a basic solvent.

In one embodiment, the acylating step solvent is pyridine.

In one embodiment, the acylating step solvent isN,N-diisopropylethylamine.

In one embodiment, the acylating step conditions include a reactiontemperature for a reaction time.

In one embodiment, the reaction temperature is about 90-150° C.

In one embodiment, the reaction temperature is about 110-130° C.

In one embodiment, the reaction temperature is about 120° C.

In one embodiment, the reaction temperature is about 80-110° C.

In one embodiment, the reaction temperature is about 80-100° C.

In one embodiment, the reaction temperature is about 90° C.

In one embodiment, the reaction time is about 30 minutes to 30 hours.

In one embodiment, the reaction time is about 12 hours to 24 hours.

In one embodiment, the reaction time is about 12 hours to 18 hours.

In one embodiment, the reaction time is about 18 hours.

In one embodiment, the reaction time is about 30 minutes to 4 hours.

In one embodiment, the reaction time is about 1 hour to 3 hours.

In one embodiment, the reaction time is about 2 hours.

For example, in one embodiment, the reduced compound (˜20 mmol), addedacetic anhydride (˜0.40 cm³) and pyridine (˜10 cm³) are combined, andthe resulting mixtures is heated at ˜120° C. for ˜18 hours withstirring. The mixture is then cooled and then poured carefully overice-water while stirring to give a solid, which is filtered, washed withwater (˜100 cm³), and dried in an oven at ˜60° C. to give the desiredacetylated reagent compound.

For example, in one embodiment, the reduced compound (˜20 mmol), addedacetic anhydride (˜25 cm³), and N,N-diisopropylethylamine (˜9 cm³) arecombined, and the resulting mixtures is heated at ˜90° C. for ˜2 hourswith stirring. The mixture is then cooled and had water (50 cm³) addedwhile stirring to give a solid, which is filtered, washed with water(4×6 cm³), and dried in an oven at ˜60° C. to give the desiredacetylated reagent compound.

The Purification (PUR) Step

The purification (PUR) step may be performed using any suitable means ofpurification.

In one embodiment, the purification (PUR) step comprises precipitation(e.g., of a reaction product) to form a precipitate, followed bycollection of the precipitate (e.g., by filtration).

Optionally, the purification (PUR) step further comprises the subsequentstep of washing of the precipitate one or more (e.g., 1, 2, 3, 4) times,for example, with a suitable washing solvent.

In one embodiment, the purification (PUR) step comprises precipitation(e.g., of a reaction product), followed by collection of the precipitate(e.g., by filtration), followed by washing of the precipitate one ormore (e.g., 1, 2, 3, 4) times, for example, with a suitable washingsolvent.

Optionally, the purification further comprises, after collecting theprecipitate and/or after washing the precipitate, a step of drying theprecipitate or the washed precipitate, for example, drying in an ovenand/or drying under vacuum.

In one embodiment, the purification (PUR) step comprises precipitation(e.g., of a reaction product), collection of the precipitate (e.g., byfiltration), followed by drying of the washed precipitate.

In one embodiment, the purification (PUR) step comprises precipitation(e.g., of a reaction product), collection of the precipitate (e.g., byfiltration), followed by washing of the precipitate one or more (e.g.,1, 2, 3, 4) times, for example, with a suitable washing solvent,followed by drying of the washed precipitate.

In one embodiment, the purification (PUR) step comprises, or furthercomprises, recrystallisation.

In one embodiment, the recrystallisation comprises: adding the compoundto a suitable solvent; heating the mixture to dissolve (preferably fullydissolve) the compound; cooling the heated mixture or allowing theheated mixture to cool so as to allow the compound to precipitate; andcollecting the precipitate (e.g., by filtration).

Optionally, the recrystallisation further comprises the subsequent stepof washing the precipitate one or more (e.g., 1, 2, 3, 4) times, forexample, with a suitable washing solvent, for example, the same solventused to dissolve the compound.

For example, in one embodiment, the recrystallisation is performed byadding the compound (e.g., 3,7-dimethylamino-10-acetyl-phenothiazine)(˜20 mmol) to ethanol (˜25 cm³), heating the mixture to ˜78° C., coolingthe heated mixture to ˜5° C. so as to allow the compound to precipitate,and filtering the mixture to collect the precipitate. The precipitate isthen washed with ethanol (e.g., 3×6 cm³). The washed precipitate is thendried in an oven at ˜60° C. for ˜3 hours.

In one embodiment, the purification (PUR) step is, or further comprises,a step of treatment with activated charcoal (also known as activatedcarbon); for example, adding activated charcoal, followed by filteringto remove the charcoal. This step may be performed, for example, using asolution of the compound in a suitable solvent.

In one embodiment, the step of treatment with activated charcoalcomprises: adding the compound to a suitable solvent to dissolve(preferably fully dissolve) the compound; adding activated charcoal tothe mixture; filtering the mixture to remove the charcoal.

The resulting filtrate may be used in a subsequent step, for example,the deacylation (DAC) step.

Optionally, this step of treatment with activated charcoal may beperformed, for example, in combination with recrystallisation.

For example, in one embodiment, the purification (PUR) step is, orcomprises: adding the compound to a suitable solvent; heating themixture to dissolve (preferably fully dissolve) the compound; addingactivated charcoal to the mixture; filtering the mixture to remove thecharcoal; cooling the heated mixture or allowing the heated mixture tocool so as to allow the compound to precipitate; and collecting theprecipitate (e.g., by filtration).

For example, in one embodiment, the recrystallisation is performed byadding the compound (e.g., 3,7-dimethylamino-10-acetyl-phenothiazine)(˜20 mmol) to ethanol (˜100 cm³), heating the mixture to ˜78° C. untilall of the compound has dissolved, adding activated charcoal (˜1 g),filtering to remove the charcoal; cooling the heated mixture to ˜5° C.so as to allow the compound to precipitate, and filtering the mixture tocollect the precipitate. The precipitate is then washed with ethanol(e.g., once, with ˜20 cm³). The washed precipitate is then dried in anoven at ˜60° C. for ˜3 hours.

The Converting (CON) Step

The converting (CON) step may be performed using any suitable reagentsand/or conditions.

In one embodiment, the converting (CON) step is by reaction with one ormore converting reagents, under converting step conditions.

In one embodiment, the converting (CON) step comprises (i) a nitroreduction step and (ii) a subsequent amino alkylation step.

For example, in one embodiment, the step of: converting (CON)3,7-dinitro-10-acetyl-phenothiazine to3,7-di(dimethylamino)-10-acetyl-phenothiazine comprises the steps of:

(i) reducing 3,7-dinitro-10-acetyl-phenothiazine to give3,7-diamino-10-acetyl-phenothiazine and subsequently

(ii) methylating 3,7-diamino-10-acetyl-phenothiazine to give3,7-di(dimethylamino)-10-acetyl-phenothiazine.

In one embodiment, the nitro reduction step is by reaction with one ormore nitro reduction reagents, under nitro reduction step conditions.

In one embodiment, the one or more nitro reduction reagents includepalladium.

In one embodiment, the one or more nitro reduction reagents includepalladium and hydrogen.

In one embodiment, the nitro reduction step is by reaction withpalladium and hydrogen.

In one embodiment, the nitro reduction step conditions include areaction temperature for a reaction time.

In one embodiment, the reaction temperature is about 40-100° C.

In one embodiment, the reaction temperature is about 50-70° C.

In one embodiment, the reaction temperature is about 60° C.

In one embodiment, the reaction time is about 1 to 36 hours.

In one embodiment, the reaction time is about 12 to 24 hours.

In one embodiment, the reaction time is about 18 hours.

In one embodiment, the nitro reduction step conditions include the useof a nitro reduction step solvent.

In one embodiment, the nitro reduction step solvent is tetrahydrofuran.

For example, in one embodiment, a mixture of acylated upstream precursor(e.g., 3,7-dinitro-10-acetyl-phenothiazine) (˜6 mmol), palladium 10% ondry carbon (˜0.2 g) and tetrahydrofuran (˜20 cm³) is heated to 60° C.under an atmosphere of hydrogen and stirred at this temperature for ˜18hours. The mixture is cooled to room temperature, poured over celitefilter aid, and washed with tetrahydrofuran (˜10 cm³). The THF filtrateis acidified with hydrochloric acid (˜0.10 M, ˜4 cm³) to precipitate theproduct as a solid. The suspension is filtered to give the desiredcompound, which is dried at ˜60° C. for ˜3 hours.

In one embodiment, the amino alkylation step is by reaction with one ormore amino alkylation reagents, under amino alkylation step conditions.

In one embodiment, the one or more amino alkylation reagents includesodium cyano borohydride (NaCNBH₃) and paraformaldehyde ((H₂CO)_(n)).

In one embodiment, the amino alkylation step is by reaction with sodiumcyano borohydride (NaCNBH₃) and paraformaldehyde ((H₂CO)_(n)).

In one embodiment, the amino alkylation step conditions include areaction temperature for a reaction time.

In one embodiment, the reaction temperature is about 20-80° C.

In one embodiment, the reaction temperature is about 30-70° C.

In one embodiment, the reaction temperature is about 50° C.

In one embodiment, the reaction time is about 10 minutes to 6 hours.

In one embodiment, the reaction time is about 1 to 3 hours.

In one embodiment, the reaction time is about 2 hours.

In one embodiment, the amino alkylation step conditions include the useof an amino alkylation step solvent.

In one embodiment, the amino alkylation step solvent is acetic acid.

For example, in one embodiment, an acid salt of a nitro-reduced acylatedupstream precursor (e.g., 3,7-diamino-10-acetyl-phenothiazinedihydrochloride) (˜7.5 mmol) is dissolved in water and sodium hydroxidesolution added to obtain a precipitate. The solid is filtered to givethe free amine, which is dissolved in acetic acid (˜20 cm³), andp-formaldehyde (˜150 mmol) and sodium cyanoborohydride (˜75 mmol) isadded. The mixture is stirred at ˜50° C. for ˜2 hours, after which water(˜50 cm³) is added and the solid is filtered to give crude product,which is crystallized from ethanol.

The Deacylating (DAC) Step

The deacylating (DAC) step may be performed using any suitabledeacylating reagents and/or conditions.

In one embodiment, the deacylating (DAC) step is by reaction with one ormore deacylating reagents, under deacylating step conditions.

In one embodiment, the one or more deacylating reagents include aBronsted acid.

In one embodiment, the one or more deacylating reagents include aninorganic Bronsted acid.

In one embodiment, the one or more deacylating reagents include ahydrohalic acid, for example, hydrochloric acid (HCl), hydrobromic acid(HBr), or hydroiodic acid (HI).

In one embodiment, the one or more deacylating reagents includehydrochloric acid.

For example, in one embodiment, the deacylating step is by reaction withhydrochloric acid (HCl).

In one embodiment, the deacylation step conditions include a reactiontemperature for a reaction time.

In one embodiment, the reaction temperature is about 60-100° C.

In one embodiment, the reaction temperature is about 70-90° C.

In one embodiment, the reaction temperature is about 80° C.

In one embodiment, the reaction time is about 10 minutes to 6 hours.

In one embodiment, the reaction time is about 20 minutes to 3 hours.

In one embodiment, the reaction time is about 1 hour.

In one embodiment, the deacylating step conditions include the use of adeacylating step solvent.

In one embodiment, the deacylating step solvent is water.

For example, in one embodiment, the acylated reagent compound (˜3 mmol),water (˜10 cm³), and hydrochloric acid (˜10 M, ˜3 cm³) are combined, andthe resulting mixture is heated at ˜80° C. for ˜1 hour with stirring.The resulting reaction product mixture contains the deacylated product.

For example, in one embodiment, the acylated reagent compound (˜20mmol), water (˜0.14 cm³), and hydrochloric acid (˜10 M, ˜6.6 cm³) arecombined, and the resulting solution is treated with activated charcoal(1 g), filtered, and the reaction mixture is heated at ˜80° C. for ˜1hour with stirring. The resulting reaction product mixture contains thedeacylated product.

The Oxidizing (OX) Step

The oxidizing (OX) step may be performed using any suitable oxidizingreagents and/or conditions.

In one embodiment, the oxidizing (OX) step is by reaction with one ormore oxidizing reagents, under oxidizing step conditions.

In one embodiment, the one or more oxidizing reagents include a Lewisacid.

In one embodiment, the one or more oxidizing reagents include FeCl₃,provided for example, as a hydrate, for example, as FeCl₃.6H₂O.

In one embodiment, the one or more oxidizing reagents include a nitrite.

In one embodiment, the one or more oxidizing reagents include aC₁₋₆alkyl nitrite.

In one embodiment, the one or more oxidizing reagents include isoamylnitrite.

In one embodiment, the one or more oxidizing reagents include t-butylnitrite.

In one embodiment, the one or more oxidizing reagents include anAmberlite resin I. R. 120 (an anion exchange resin), which, in thepresent case, acts as an oxidizing agent.

In one embodiment, the oxidizing step conditions include a reactiontemperature for a reaction time.

In one embodiment, the reaction temperature is about 1-25° C.

In one embodiment, the reaction temperature is about 1-15° C.

In one embodiment, the reaction temperature is about 1-10° C.

In one embodiment, the reaction temperature is about 1-10° C.

In one embodiment, the reaction temperature is about 2-10° C.

In one embodiment, the reaction temperature is about 1-9° C.

In one embodiment, the reaction temperature is about 2-9° C.

In one embodiment, the reaction temperature is about 1-8° C.

In one embodiment, the reaction temperature is about 2-8° C.

In one embodiment, the reaction temperature is about 1-7° C.

In one embodiment, the reaction temperature is about 2-7° C.

In one embodiment, the reaction temperature is about 1-6° C.

In one embodiment, the reaction temperature is about 2-6° C.

In one embodiment, the reaction temperature is about 1-5° C.

In one embodiment, the reaction temperature is about 2-5° C.

In one embodiment, the reaction temperature is about 5° C.

Without wishing to be bound by any particular theory, the inventorsbelieve that by employing a relatively low oxidizing step temperature(e.g., below ˜10° C.; e.g., below ˜5° C.), the production of undesiredby-products (including, e.g., the reintroduction of Azure B, etc.) canbe minimized and the purity of the final product can be maximized.

In one embodiment, the reaction time is about 5 minutes to 3 hours.

In one embodiment, the reaction time is about 15 minutes to 2 hours.

In one embodiment, the reaction time is about 30 minutes.

In one embodiment, the oxidizing step conditions include the use of anoxidizing step solvent.

In one embodiment, the oxidizing step solvent is water.

For example, in one embodiment, the reaction product mixture obtained bythe deacylating (DAC) step and containing the deacylated product (˜3mmol) is cooled to ˜5° C., and a cooled aqueous solution of FeCl₃ (˜3mmol FeCl₃.6H₂O in ˜10 cm³ water, at ˜5° C.) is added. The resultingmixture is held at ˜5° C. for ˜30 minutes with stirring. The resultingreaction product mixture contains the diaminophenothiazinium compound.

For example, in one embodiment, the reaction product mixture obtained bythe deacylating (DAC) step and containing the deacylated product (˜20mmol) is cooled to ˜5° C., and a cooled aqueous solution of FeCl₃ (˜40mmol FeCl₃.6H₂O in ˜80 cm³ water, at ˜5° C.) is added. The resultingmixture is held at ˜5° C. for ˜30 minutes with stirring. The resultingreaction product mixture contains the diaminophenothiazinium compound.

Optional Additional Purification Steps

Each of the optional additional purification steps (PUR^(RED-AC),PUR^(DA-OX), PUR^(OX), and PUR^(AC2-CON)), if present, may be performedusing any suitable means of purification.

In one embodiment, one or more or all of the optional additionalpurification steps (PUR^(RED-AC), PUR^(DA-OX), PUR^(OX), PUR^(AC-CON)),if present, is as defined above under the heading “The Purification(PUR) Step.”

In one embodiment, one or both of the optional additional purificationsteps PUR^(RED-AC) and PUR^(DA-OX), if present, is as defined aboveunder the heading “The Purification (PUR) Step.”

Optional Additional Purification: PUR^(OX)

In one embodiment, after said oxidizing (OX) step, saiddiaminophenothiazinium compound obtained in said oxidizing (OX) step ispurified (PUR^(OX)).

In one embodiment, the purifying (PUR^(OX)) step is, or comprises,recrystallisation.

In one embodiment, the recrystallisation comprises adding the compoundto a suitable solvent (e.g., water); heating the mixture to dissolve(preferably fully dissolve) the compound; cooling the heated mixture orallowing the heated mixture to cool so as to allow the compound toprecipitate; and collection of the precipitate (e.g., by filtration).

In one embodiment, the recrystallisation includes a step of adjustingthe pH of the mixture of compound and suitable solvent (e.g., water) tobe about 0.5 to about 2.5 (e.g., about 1 to about 2), for example, usingHCl. This additional step may be performed, for example, before coolingthe heated mixture or allowing the heated mixture to cool, or, morepreferably, before heating the mixture to dissolve the compound.

In one embodiment, the recrystallisation comprises adding the compoundto a suitable solvent (e.g., water); adjusting the pH of the mixture tobe about 0.5 to about 2.5 (e.g., about 1 to about 2) using HCl; heatingthe mixture to dissolve (preferably fully dissolve) the compound;cooling the heated mixture or allowing the heated mixture to cool so asto allow the compound to precipitate; and collection of the precipitate(e.g., by filtration).

Optionally, the recrystallisation further comprises the subsequent stepof washing of the precipitate one or more (e.g., 1, 2, 3, 4) times, forexample, with a suitable washing solvent, for example, the same solventused to dissolve the compound.

Optionally, the recrystallisation further comprises, after collection ofthe precipitate and/or after washing of the precipitate, a step ofdrying the precipitate or washed precipitate, for example, drying in anoven and/or drying under vacuum.

For example, in one embodiment, the recrystallisation is performed byadding the compound (e.g., methylthioninium chloride) (˜1 g, ˜3 mmol) towater (˜40 cm³), adjusting the pH of the mixture to be ˜1.7 usingaqueous hydrochloric acid (HCl, 5 M), heating the mixture to ˜80° C.until all of the compound has dissolved, allowing the mixture to coolnaturally to ˜25° C. while stirring so as to allow the compound toprecipitate, filtering the mixture to collect the precipitate, anddrying in an oven at ˜60° C. for ˜18 hours.

For example, in one embodiment, the recrystallisation is performed byadding the compound (e.g., methylthioninium chloride) (˜1 g, ˜3 mmol) towater (˜20 cm³), adjusting the pH of the mixture to be ˜1 to ˜2 usingaqueous hydrochloric acid (HCl, 10 M, 0.33 cm³), heating the mixture to˜80° C. until all of the compound has dissolved, allowing the mixture tocool naturally to ˜25° C. while stirring so as to allow the compound toprecipitate, filtering the mixture to collect the precipitate, anddrying in an oven at ˜60° C. for ˜18 hours.

Purity

The methods described herein yield diaminophenothiazinium compounds asdefined herein and including, for example, methylthioninium chloride(MTC), at a purity that, until now, has been unavailable worldwide.

For example, many of the methods described herein yield very high purityMTC with extremely low levels of both organic impurities (e.g., of AzureB, Azure A, Azure C, and Methylene Violet Bernthsen (MVB)) and metalimpurities (e.g., meeting or exceeding the European Pharmacopoeia (EP)limits).

Thus, one aspect of the present invention pertains to adiaminophenothiazinium compound as defined herein and including, forexample, methylthioninium chloride (MTC), that has a purity as definedherein.

In one embodiment, the present invention pertains to methylthioniniumchloride (MTC) that has a purity as defined herein.

Another aspect of the present invention pertains to adiaminophenothiazinium compound as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and that has a purity as defined herein.

In one embodiment, the present invention pertains to methylthioniniumchloride (MTC) that is obtained by, or is obtainable by, a method ofsynthesis and/or purification as described herein, and that has a purityas defined herein.

In one embodiment, the compound (e.g., MTC) has a purity of 99.7% orgreater.

In one embodiment, the compound (e.g., MTC) has a purity of 99.6% orgreater.

In one embodiment, the compound (e.g., MTC) has a purity of 99.5% orgreater.

In one embodiment, the compound (e.g., MTC) has a purity of 99% orgreater.

In one embodiment, the compound (e.g., MTC) has a purity of 98% orgreater.

In one embodiment, the compound has less than 0.1% Azure B as impurity.

In one embodiment, the compound has less than 0.5% Azure B as impurity.

In one embodiment, the compound has less than 1% Azure B as impurity.

In one embodiment, the compound has less than 2% Azure B as impurity.

In one embodiment, the compound has less than 0.05% Azure A as impurity.

In one embodiment, the compound has less than 0.10% Azure A as impurity.

In one embodiment, the compound has less than 0.15% Azure A as impurity.

In one embodiment, the compound has less than 0.05% Azure C as impurity.

In one embodiment, the compound has less than 0.10% Azure C as impurity.

In one embodiment, the compound has less than 0.15% Azure C as impurity.

In one embodiment, the compound has less than 0.02% MVB as impurity.

In one embodiment, the compound has less than 0.05% MVB as impurity.

(All percentage purities recited herein are weight/weight unlessotherwise specified.)

In one embodiment, the compound (e.g., MTC) has an elementals purity(e.g., for each of Al, Cr, Zn, Cu, Fe, Mn, Hg, Ni, Mo, Cd, Sn, and Pb)that is equal to or better than the values mentioned in column “VersionEP4” in Table 1 below (believed to be the European Pharmacopoeia (EP)limits for Version EP4 set in 2002).

In one embodiment, the compound (e.g., MTC) has an elementals purity(e.g., for each of Al, Cr, Zn, Cu, Fe, Mn, Hg, Ni, Mo, Cd, Sn, and Pb)that is equal to or better than the values mentioned in column “VersionEP5.4” in Table 1 below (believed to be the European Pharmacopoeia (EP)limits for Version EP5.4 set in 2006).

The term “elementals purity” referred to herein pertains to the amountsof the twelve (12) metals specified by the European Pharmacopoeia: Al,Cr, Zn, Cu, Fe, Mn, Hg, Ni, Mo, Cd, Sn, and Pb.

TABLE 1 Elementals Purity (μg/g) Element Version EP4 Version EP5.4Aluminium (Al) 100 100 Cadmium (Cd) 1 1 Chromium (Cr) 10 100 Copper (Cu)100 300 Tin (Sn) 10 10 Iron (Fe) 100 200 Manganese (Mn) 10 10 Mercury(Hg) 1 1 Molybdenum (Mo) 10 10 Nickel (Ni) 10 10 Lead (Pb) 10 10 Zinc(Zn) 10 100

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.9 times the values quoted for “VersionEP4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.9 times the values quoted for “VersionEP5.4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.8 times the values quoted for “VersionEP4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.8 times the values quoted for “VersionEP5.4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.7 times the values quoted for “VersionEP4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.7 times the values quoted for “VersionEP5.4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.5 times the values quoted for “VersionEP4” in Table 1.

In one embodiment, the compound (e.g., MTC) has an elementals puritythat is equal to or better than 0.5 times the values quoted for “VersionEP5.4” in Table 1.

(For example, 0.5 times the values quoted for “Version EP4” in Table 1are 50 μg/g Al, 0.5 μg/g Cd, 5 μg/g Cr, etc.)

In one embodiment, the compound (e.g., MTC) has a chromium purity thatis equal to or better than 10 μg/g.

In one embodiment, the compound (e.g., MTC) has a chromium purity thatis equal to or better than 100 μg/g.

In one embodiment, the compound (e.g., MTC) has a copper purity that isequal to or better than 10 μg/g.

In one embodiment, the compound (e.g., MTC) has an iron purity that isequal to or better than 100 μg/g.

All plausible and compatible combinations of the above purity grades aredisclosed herein as if each individual combination was specifically andexplicitly recited.

Compositions

One aspect of the present invention pertains to a composition comprisinga diaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein.

One aspect of the present invention pertains to a composition comprisinga diaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein.

One aspect of the present invention pertains to a composition comprisinga diaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that has a purity as definedherein.

One aspect of the present invention pertains to a composition comprisinga diaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and that has a purity as defined herein.

In one embodiment, the composition is a pharmaceutical composition.

In one embodiment, the pharmaceutical composition further comprises apharmaceutically acceptable carrier, diluent, or excipient.

Compositions and formulations are discussed in more detail below.

Methods of Inactivating Pathogens

One aspect of the present invention pertains to the use of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein, in a method ofinactivating a pathogen in a sample (for example a blood or plasmasample), for example, in vitro, the method comprising the steps ofintroducing the compound into the sample, and subsequently exposing thesample to light.

Another aspect of the present invention pertains to a method ofinactivating a pathogen in a sample, for example, in vitro, comprisingthe steps of introducing an effective amount of a compound into thesample, and subsequently exposing the sample to light, wherein thecompound is a diaminophenothiazinium compound, as defined herein andincluding, for example, methylthioninium chloride (MTC), that isobtained by, or is obtainable by, a method of synthesis and/orpurification as described herein, and/or that has a purity as definedherein.

Use in Methods of Medical Treatment

One aspect of the present invention pertains to a diaminophenothiaziniumcompound, as defined herein and including, for example, methylthioniniumchloride (MTC), that is obtained by, or is obtainable by, a method ofsynthesis and/or purification as described herein, and/or that has apurity as defined herein, for use in a method of treatment (e.g., amethod of treatment or prophylaxis, e.g., a method of treatment orprophylaxis of a disease condition, as described herein) of the human oranimal body by therapy.

One aspect of the present invention pertains to a diaminophenothiaziniumcompound, as defined herein and including, for example, methylthioniniumchloride (MTC), that is obtained by, or is obtainable by, a method ofsynthesis and/or purification as described herein, for use in a methodof treatment (e.g., a method of treatment or prophylaxis of a diseasecondition, as described herein) of the human or animal body by therapy.

One aspect of the present invention pertains to a diaminophenothiaziniumcompound, as defined herein and including, for example, methylthioniniumchloride (MTC), that has a purity as defined herein, for use in a methodof treatment (e.g., a method of treatment or prophylaxis of a diseasecondition, as described herein) of the human or animal body by therapy.

One aspect of the present invention pertains to a diaminophenothiaziniumcompound, as defined herein and including, for example, methylthioniniumchloride (MTC), that is obtained by, or is obtainable by, a method ofsynthesis and/or purification as described herein, and that has a purityas defined herein, for use in a method of treatment (e.g., a method oftreatment or prophylaxis of a disease condition, as described herein) ofthe human or animal body by therapy.

Use in the Manufacture of Medicaments

One aspect of the present invention pertains to the use of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein, in the manufactureof a medicament for use in the treatment or prophylaxis of a diseasecondition, as described herein.

One aspect of the present invention pertains to the use of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, in the manufacture of a medicament for use in the treatment orprophylaxis of a disease condition, as described herein.

One aspect of the present invention pertains to the use of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and that has a purity as defined herein, in the manufacture of amedicament for use in the treatment or prophylaxis of a diseasecondition, as described herein.

Methods of Treatment

One aspect of the present invention pertains to a method of treatment orprophylaxis of a disease condition, as described herein, in a patient,comprising administering to said patient a therapeutically-effectiveamount or a prophylactically-effective amount of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and/or that has a purity as defined herein.

One aspect of the present invention pertains to a method of treatment orprophylaxis of a disease condition, as described herein, in a patient,comprising administering to said patient a therapeutically-effectiveamount or a prophylactically-effective amount of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein.

One aspect of the present invention pertains to a method of treatment orprophylaxis of a disease condition, as described herein, in a patient,comprising administering to said patient a therapeutically-effectiveamount or a prophylactically-effective amount of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that has a purity as definedherein.

One aspect of the present invention pertains to a method of treatment orprophylaxis of a disease condition, as described herein, in a patient,comprising administering to said patient a therapeutically-effectiveamount or a prophylactically-effective amount of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and that has a purity as defined herein.

One aspect of the present invention pertains to a method of regulatingthe aggregation of a tau protein in the brain of a mammal, whichaggregation is associated with a disease statem as described herein, thetreatment comprising administering to said mammal in need of saidtreatment, a prophylactically- or therapeutically-effective amount of aninhibitor of said aggregation, wherein the inhibitor is adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method of synthesis and/or purification as describedherein, and that has a purity as defined herein.

Disease Conditions

In one embodiment, the disease condition is a tauopathy.

In one embodiment, the disease condition is a disease of tau proteinaggregation.

Diseases which are characterized primarily or partially by abnormal tauaggregation are referred to herein as “tauopathies” or “diseases of tauprotein aggregation.” Examples of such diseases are discussed in thearticle by Wischik et al. in Neurobiology of Alzheimer's Disease, 2ndEdition, 2000, Eds. Dawbarn, D. and Allen, S. J., The Molecular andCellular Neurobiology Series, Bios Scientific Publishers, Oxford, UK.

In one embodiment, the disease condition is Alzheimer's disease (AD);Pick's disease; Progressive Supranuclear Palsy (PSP); fronto-temporaldementia (FTD); FTD and parkinsonism linked to chromosome 17 (FTDP-17);disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;pallido-nigro-luysian degeneration (PNLD); or cortico-basal degeneration(CBD).

In one embodiment, the disease condition is Alzheimer's disease (AD).

In one embodiment, the disease is a disease of tau protein aggregation,as described herein, and the effective amount is an amount sufficient toinhibit the aggregation of the tau protein associated with said diseasestate.

In one embodiment, the disease condition is mild cognitive impairment(MCI).

In one embodiment, the disease condition is skin cancer.

In one embodiment, the disease condition is melanoma.

In one embodiment, the disease condition is methemoglobinemia.

In one embodiment, the disease condition is a viral, bacterial,protozoal, or parasitic disease condition (e.g., a viral infection, abacterial infection, a protozoal infection, a paraisitic infection).

In one embodiment, the disease condition is a viral infection.

In one embodiment, the disease condition is a bacterial infection.

In one embodiment, the disease condition is a protozoal infection.

In one embodiment, the disease condition is a parasitic infection.

In one embodiment, the disease condition is parasitic infection with aparasite of Trypanosoma, Leishmania, Eimeria, Neospora, Cyclospora, orCryptosporidia family.

In one embodiment, the disease condition is infection with Plasmodiumvivax, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale,Trypanosoma protoza, Entamoeba histolytica, Trichomonas vaginalis,Giardia larnblia, Trypanosoma brucei gambiense, Trypanosoma bruceirhodesiense, Trypanosoma cruzi, Leishmania major, Leishmania tropica,Leishmania aethiopica, Leishmania infantum, Leishmania braiiliensis,Leishmania mexicana, Leishmania arnazonensis, Leishmaniadonovani-Leishmania infantum complex, Cryptosporidiurn parvum,Toxoplasma gondii, Encephalitozoon species, Nosema species, or Septataintestinalis.

In one embodiment, the disease condition is malaria, visceral

leishmaniasis (often known as kalaazar), African sleeping sickness,

toxoplasmosis, giardiasis, or Chagas' disease.

In one embodiment, the disease condition is malaria (i.e., an example ofa protozoal disease condition).

In this embodiment (i.e., the disease condition is malaria), thetreatment may be in combination with one or more other antimicrobialagents, for example, one or more of chloroquine, atovaquone, quinine,primethamine, sulfadiazine, and primaquine.

In one embodiment, the disease condition is, or is caused by, HepatitisC virus (HCV), human immunodeficiency virus (HIV), or West Nile virus(WNV).

In one embodiment, the disease condition is Hepatitis C virus (HCV)infection.

In one embodiment, the disease condition is human immunodeficiency virus(HIV) infection.

In one embodiment, the disease condition is West Nile virus (WNV)infection.

In one embodiment, the disease condition is a synucleinopathy.

As those skilled in the art will be aware, the term synucleinopathies isused to name a group of neurodegenerative disorders characterized byfibrillary aggregates of synuclein protein, particularly α-synuclein, inthe cytoplasm of selective populations of neurons and glia, and inparticular in which the presence of synuclein-containing inclusions arepathognomic for the disease. This should be distinguished fromnon-synucleinopathy disorders in which synuclein-containing inclusionsmay or may not be present in addition to other pathologies.

The synucleinopathies currently consist of the following disorders:Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiplesystem atrophy (MSA), drug-induced parkinsonism (e.g. produced by1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or pesticides suchas rotenone), and pure autonomic failure (PAF).

In one embodiment, the disease condition is Parkinson's disease (PD).

In one embodiment, the disease condition is dementia with Lewy bodies(DLB).

In one embodiment, the disease condition is multiple system atrophy(MSA).

In one embodiment, the disease condition is drug-induced parkinsonism.

In one embodiment, the disease condition is pure autonomic failure(PAF).

Treatment

The term “treatment,” as used herein in the context of treating adisease condition, pertains generally to treatment and therapy, whetherof a human or an animal (e.g., in veterinary applications), in whichsome desired therapeutic effect is achieved, for example, the inhibitionof the progress of the condition, and includes a reduction in the rateof progress, a halt in the rate of progress, regression of thecondition, amelioration of the condition, and cure of the condition.Treatment as a prophylactic measure (i.e., prophylaxis, prevention) isalso included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired prophylactic effect, commensuratewith a reasonable benefit/risk ratio, when administered in accordancewith a desired treatment regimen.

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g., drugs, antibodies (e.g., as inimmunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT,ADEPT, etc.); surgery; radiation therapy; and gene therapy.

Routes of Administration

The diaminophenothiazinium compound, or pharmaceutical compositioncomprising it, may be administered to a subject/patient by anyconvenient route of administration, whether systemically/peripherally ortopically (i.e., at the site of desired action).

Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal (including, e.g., intracatheter injection into the brain);by implant of a depot or reservoir, for example, subcutaneously orintramuscularly.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

Suitable subjects for the methods involving Alzheimer's disease may beselected on the basis of conventional factors. Thus, the initialselection of a patient may involve any one or more of: rigorousevaluation by an experienced clinician; exclusion of non-Alzheimer'sdisease diagnosis as far as possible by supplementary laboratory andother investigations; and objective evaluation of level of cognitivefunction using neuropathologically validated battery.

In one embodiment, the subject/patient is not a human.

Formulations

While it is possible for the diaminophenothiazinium compound to be used(e.g., administered) alone, it is often preferable to present it as acomposition or formulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising adiaminophenothiazinium compound, as described herein, and apharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one diaminophenothiazinium compound, as describedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, including, but notlimited to, pharmaceutically acceptable carriers, diluents, excipients,adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants,stabilisers, solubilisers, surfactants (e.g., wetting agents), maskingagents, colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, New York, USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least onediaminophenothiazinium compound, as defined herein, together with one ormore other pharmaceutically acceptable ingredients well known to thoseskilled in the art, e.g., carriers, diluents, excipients, etc. Ifformulated as discrete units (e.g., tablets, etc.), each unit contains apredetermined amount (dosage) of the active compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the active ingredient in the liquid is from about 1ng/mL to about 10 μg/mL, for example from about 10 ng/mL to about 1μg/mL. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

Examples of Preferred Formulations

One aspect of the present invention pertains to a dosage unit (e.g., apharmaceutical tablet or capsule) comprising 20 to 300 mg of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method as described herein, and/or that has a purity asdefined herein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

One aspect of the present invention pertains to a dosage unit (e.g., apharmaceutical tablet or capsule) comprising 20 to 300 mg of adiaminophenothiazinium compound, as defined herein and including, forexample, methylthioninium chloride (MTC), that is obtained by, or isobtainable by, a method as described herein, and/or that has a purity asdefined herein.

In one embodiment, the dosage unit is a tablet.

In one embodiment, the dosage unit is a capsule.

In one embodiment, the amount is 30 to 200 mg.

In one embodiment, the amount is about 25 mg.

In one embodiment, the amount is about 30 mg.

In one embodiment, the amount is about 35 mg.

In one embodiment, the amount is about 50 mg.

In one embodiment, the amount is about 60 mg.

In one embodiment, the amount is about 70 mg.

In one embodiment, the amount is about 100 mg.

In one embodiment, the amount is about 125 mg.

In one embodiment, the amount is about 150 mg.

In one embodiment, the amount is about 175 mg.

In one embodiment, the amount is about 200 mg.

In one embodiment, the amount is about 250 mg.

In one embodiment, the dosage unit further comprises a pharmaceuticallyacceptable carrier, diluent, or excipient.

In one embodiment, the pharmaceutically acceptable carrier, diluent, orexcipient is or comprises one or both of a glyceride (e.g., Gelucire44/14®; lauroyl macrogol-32 glycerides PhEur, USP) and colloidal silicondioxide (e.g., 2% Aerosil 200®; Colliodal Silicon Dioxide PhEur, USP).

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the diaminophenothiazinium compound, and compositionscomprising the diaminophenothiazinium compound, can vary from patient topatient. Determining the optimal dosage will generally involve thebalancing of the level of therapeutic benefit against any risk ordeleterious side effects. The selected dosage level will depend on avariety of factors including, but not limited to, the activity of theparticular compound, the route of administration, the time ofadministration, the rate of excretion of the compound, the duration ofthe treatment, other drugs, compounds, and/or materials used incombination, the severity of the condition, and the species, sex, age,weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the diaminophenothiazinium compound is inthe range of about 100 ng to about 25 mg (more typically about 1 to 10mg) per kilogram body weight of the subject per day. Where thediaminophenothiazinium compound is a salt, an ester, an amide, aprodrug, or the like, the amount administered is calculated on the basisof the parent compound and so the actual weight to be used is increasedproportionately.

In one embodiment, the diaminophenothiazinium compound (e.g., MTC) isadministered to a human patient according to one of the following dosageregimes:

about 50 mg, 3 times daily;

about 50 mg, 4 times daily;

about 75 mg, 3 times daily;

about 75 mg, 4 times daily;

about 100 mg, 2 times daily;

about 100 mg, 3 times daily;

about 125 mg, 2 times daily;

about 125 mg, 3 times daily;

about 150 mg, 2 times daily;

about 200 mg, 2 times daily.

Combination Treatments and Therapies

Any of the medical uses or methods described herein may be used as partof a combination treatment or therapy, that is, a treatment or therapyin which two or more treatments or therapies are combined, for example,sequentially or simultaneously.

In one embodiment, a treatment (e.g., employing a diaminophenothiaziniumcompound as described herein) is in combination with one or more otherantimicrobial agents, for example, chloroquine and atovaquone.

In one embodiment, a treatment (e.g., employing a diaminophenothiaziniumcompound as described herein) is in combination with a cholinesteraseinhibitor such as Donepezil (Aricept™), Rivastigmine (Exelon™) orGalantamine (Reminyl™).

In one embodiment, a treatment (e.g., employing a diaminophenothiaziniumcompound as described herein) is in combination with an NMDA receptorantagonist such as Memantine (Ebixa™, Namenda™)

In one embodiment, a treatment (e.g. employing a diaminophenothiaziniumcompound as described herein) is in combination with a muscarinicreceptor agonist.

In one embodiment, a treatment (e.g. employing a diaminophenothiaziniumcompound as described herein) is in combination with an inhibitor ofamyloid precursor protein processing that leads to enhanced generationof beta-amyloid.

Kits

One aspect of the invention pertains to a kit comprising (a) adiaminophenothiazinium compound as described herein, or a compositioncomprising an diaminophenothiazinium compound as described herein, e.g.,preferably provided in a suitable container and/or with suitablepackaging; and (b) instructions for use, e.g., written instructions onhow to administer the compound or composition.

The written instructions may also include a list of indications forwhich the active ingredient is a suitable treatment.

For example, in one embodiment, the kit is a drug product for thetreatment of a disease in a mammal suffering therefrom, comprising acontainer labelled or accompanied by a label indicating that the drugproduct is for the treatment of said disease, the container containingone or more dosage units each comprising at least one pharmaceuticallyacceptable excipient and, as an active ingredient, adiaminophenothiazinium compound as described herein.

Ligands and Labels

The diaminophenothiazinium compounds discussed herein and that arecapable of inhibiting the aggregation of tau protein will also becapable of acting as ligands or labels of tau protein (or aggregated tauprotein). Thus, in one embodiment, the diaminophenothiazinium compoundis a ligand of tau protein (or aggregated tau protein).

Such diaminophenothiazinium compounds (ligands) may incorporate, beconjugated to, be chelated with, or otherwise be associated with, otherchemical groups, such as stable and unstable detectable isotopes,radioisotopes, positron-emitting atoms, magnetic resonance labels, dyes,fluorescent markers, antigenic groups, therapeutic moieties, or anyother moiety that may aid in a prognostic, diagnostic or therapeuticapplication.

For example, in one embodiment, the diaminophenothiazinium compound isas defined above, but with the additional limitation that the compoundincorporates, is conjugated to, is chelated with, or is otherwiseassociated with one or more (e.g., 1, 2, 3, 4, etc.) isotopes,radioisotopes, positron-emitting atoms, magnetic resonance labels, dyes,fluorescent markers, antigenic groups, or therapeutic moieties.

In one embodiment, the diaminophenothiazinium compound is a ligand aswell as a label, e.g., a label for tau protein (or aggregated tauprotein), and incorporates, is conjugated to, is chelated with, or isotherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels.

For example, in one embodiment, the diaminophenothiazinium compound isas defined above, but with the additional limitation that the compoundincorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable labels.

Labelled diaminophenothiazinium compounds (e.g., when ligated to tauprotein or aggregated tau protein) may be visualised or detected by anysuitable means, and the skilled person will appreciate that any suitabledetection means as is known in the art may be used.

For example, the diaminophenothiazinium compound (ligand-label) may besuitably detected by incorporating a positron-emitting atom (e.g., ¹¹C)(e.g., as a carbon atom of one or more alkyl group substituents, e.g.,methyl group substituents) and detecting the compound using positronemission tomography (PET) as is known in the art. Suitable methods forpreparing these and similar ¹¹C labelled diaminophenothiaziniums areshown, for example, in Wischik, C. M., et al., 2002b (see especiallyFIGS. 11a, 11b, 12 therein) and Schweiger, L. F., et al., 2005.

One aspect of the present invention pertains to a method of labellingtau protein (or aggregated tau protein) comprising the steps of:contacting the tau protein (or aggregated tau protein) with adiaminophenothiazinium compound, as described herein, that additionallyincorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable labels.

Another aspect of the present invention pertains to a method ofdetecting tau protein (or aggregated tau protein) comprising the stepsof:

(i) contacting the tau protein (or aggregated tau protein) with adiaminophenothiazinium compound, as described herein, and thatadditionally incorporates, is conjugated to, is chelated with, or isotherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)detectable labels, and

(ii) detecting the presence and/or amount of said compound bound to tauprotein (or aggregated tau protein).

Another aspect of the present invention pertains to a method ofdiagnosis or prognosis of a tauopathy in a subject believed to sufferfrom the disease, comprising the steps of:

(i) introducing into the subject a diaminophenothiazinium compoundcapable of labelling tau protein or aggregated tau protein, particularlytau protein (e.g., a diaminophenothiazinium compound, as describedherein, and that additionally incorporates, is conjugated to, ischelated with, or is otherwise associated with, one or more (e.g., 1, 2,3, 4, etc.) detectable labels),

(ii) determining the presence and/or amount of said compound bound totau protein or aggregated tau protein in the brain of the subject, and

(iii) correlating the result of the determination made in (ii) with thedisease state of the subject.

Another aspect of the present invention pertains to adiaminophenothiazinium compound capable of labelling tau protein oraggregated tau protein (e.g., a diaminophenothiazinium compound, asdescribed herein, and that additionally incorporates, is conjugated to,is chelated with, or is otherwise associated with, one or more (e.g., 1,2, 3, 4, etc.) detectable labels), for use in a method of diagnosis orprognosis of a tauopathy.

In another aspect, the present invention provides use of adiaminophenothiazinium compound capable of labelling tau protein oraggregated tau protein, particularly tau protein (e.g., adiaminophenothiazinium compound, as described herein, that additionallyincorporates, is conjugated to, is chelated with, or is otherwiseassociated with, one or more (e.g., 1, 2, 3, 4, etc.) detectablelabels), in a method of manufacture of a diagnostic or prognosticreagent for use in the diagnosis or prognosis of a tauopathy.

The diaminophenothiazinium ligands/labels may be administered directly,or they may be administered in a precursor form, for conversion to theactive form (e.g., ligating form, labelling form) by an activating agentpresent in, or administered to, the same subject.

The diaminophenothiazinium ligands/labels may be used as part of amethod of diagnosis or prognosis. They may be used to select a patientfor treatment, or to assess the effectiveness of a treatment or atherapeutic agent (e.g. an inhibitor of tau protein aggregation)administered to the subject.

EXAMPLES

The following examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

Synthesis 1 3,7-Dimethylamino-10H-phenothiazine

To a 250 cm³ round bottom flask placed under an atmosphere of argon wasadded methylthioninium chloride (Medex™) (MTC.3H₂O, MW 373.90, 10 g,26.7 mmol) and ethanol (75 cm³). Sodium borohydride (NaBH₄, MW 37.83,2.0 g, 52.9 mmol) was added in portions. The mixture was heated to 40°C. and stirred for 1 hour. The resulting yellow/green suspension wascooled to 5° C. using an ice-water bath and filtered by canular underargon, washed once with ethanol (20 cm³), and dried under vacuum at roomtemperature to give the title compound as a light green solid. Theproduct was used without further purification in Synthesis 2.

Synthesis 2 3,7-Dimethylamino-10-acetyl-phenothiazine

The 3,7-dimethylamino-10H-phenothiazine obtained in Synthesis 1 wasplaced in a 250 cm³ round bottom flask and acetic anhydride ((H₃CCO)₂O,40 cm³) and pyridine (10 cm³) were added, and the resulting solution washeated at 120° C. for 18 hours with stirring. The reaction mixture wascooled to room temperature and then poured carefully into ice-waterwhile stirring to give a light brown solid, which was filtered using astandard Buchner funnel apparatus with a water aspirator, washed oncewith water (100 cm³), and dried in an oven at 60° C. for 3 hours toyield the title compound (MW 327.45, 4.63 g, 14.1 mmol, yield 53%),which was recrystallised from ethanol by dissolving it in hot ethanol(100 cm³), adding activated charcoal (1 g), filtering to remove thecharcoal, cooling the solution to 5° C. using an ice-water bath so thata precipitate formed (colourless crystals), and filtering using astandard Buchner filter with a water aspirator in order to collect thecrystals. The crystals where then dried in an oven at 60° C. for 3hours.

δ_(H) (250 MHz; CDCl₃): 2.16 (3H, s, CH₃), 2.93 (12H, s, NCH₃),6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47(2H, brd s, ArH); δ_(C) (62.9 MHz; CDCl₃): 170.3 (C═O), 148.9 (ArC),127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7(NCH₃), 22.9 (CH₃); m/z (ES) 284.2 (100%, [M−OAc]⁺), 328.1 (15%,[M+H]⁺), 350.1 (41%, [M+Na]⁺).

Synthesis 3 Methylthioninium Chloride (MTC)

3,7-Dimethylamino-10-acetyl-phenothiazine obtained in the Synthesis 2(MW 327.45, 1 g, 3.05 mmol) was placed in a 50 cm³ round bottom flask,and water (10 cm³) and hydrochloric acid (10 M, 3 cm³) were added, andthe resulting solution was heated at 80° C. for 1 hour with stirring togive a green solution. The reaction mixture was cooled to 5° C. using anice-water bath, and cooled aqueous iron chloride hexahydrate(FeCl₃.6H₂O, MW 270.30, 0.82 g, 3.03 mmol, 10 cm³) was added, giving animmediate deep blue colour. (The aqueous iron chloride hexahydrate wasprepared by dissolving the solid iron chloride hexahydrate in water (10cm³) and cooling to 5° C. using an ice-water bath before being added tothe reaction mixture.) The reaction mixture was stirred for a further 30minutes at 5° C., filtered using a standard Buchner filter with a wateraspirator, and dried in an oven at 60° C. for 18 hours to give the titlecompound (MW 319.86, 0.51 g, 1.59 mmol, yield 52%) as green needles.

δ_(H) (250 MHz; D₂O): 6.99 (2H, d, 9.25, ArH), 6.82 (2H, d, 8, ArH),6.58 (2H, s, ArH), 2.98 (12H, s, NCH₃).

Synthesis 4 Methylthioninium Chloride (MTC)

The product was prepared as described in Synthesis 3, except that thereaction mixture was cooled to 25° C. (instead of 5° C.) before the ironchloride hexahydrate was added.

Samples of the Medex™ starting material and the products of Synthesis 3(MTC-5° C.) and Synthesis 4 (MTC-25° C.) were compared using HPLC.Chromatograms for the Medex™ starting material and MTC-5° C. are shownin FIG. 1 and FIG. 2 respectively. The impurity levels are summarised inthe following Table. Azure A, Azure, B, Azure C, and MVB (MethyleneViolet Bernthsen) are common undesirable impurities typically found insamples of MTC.

TABLE 2 MTC Azure B Azure A Azure C MVB Other Sample w/w % w/w % w/w %w/w % w/w % w/w % Medex ™ 93.76 5.46 0.18 0.23 0.09 0.28 MTC-25° C.98.98 0.92 0 0 0 0.10 MTC-5° C. 99.65 0.27 0 0 0 0.08

Synthesis 5 Methylthioninium Chloride (MTC)

3,7-Dimethylamino-10-acetyl-phenothiazine obtained in Synthesis 2 (MW327.45, 2.5 g, 7.63 mmol) was placed in a 50 cm³ round bottom flask, andwater (25 cm³) and hydrochloric acid (10 M, 7.5 cm³) were added, and theresulting solution was heated at 80° C. for 1 hour with stirring to givea green solution. The reaction mixture was cooled to 5° C. using anice-water bath, and a cooled aqueous iron chloride hexahydrate solution(FeCl₃.6H₂O, MW 270.30, 2.06 g, 7.62 mmol, 20 cm³) was added, giving animmediate deep blue colour. (The aqueous iron chloride hexahydrate wasprepared by dissolving the solid iron chloride hexahydrate in water (10cm³) and cooling to 5° C. using an ice-water bath before being added tothe reaction mixture.) The reaction mixture was stirred for a further 30minutes at 5° C., filtered using a standard Buchner filter with a wateraspirator, and dried in an oven at 60° C. for 18 hours to give the titlecompound (MW 319.86, 1.32 g, 4.12 mmol, 54%) as green needles. Thismaterial was recrystallised as described in the next synthesis.

Synthesis 6 Methylthioninium Chloride (MTC)

The product of Synthesis 5 was recrystallised by dissolving 1 g in water(40 cm³) and the pH was adjusted to 1.7 with 5 M aqueous hydrochloricacid (HCl). The suspension was then heated to 80° C. and allowed to coolto 25° C. naturally while stirring to give the highly pure titlecompound as fine green needles, which were dried in an oven at 60° C.for 18 hours (0.90 g, 90%).

δ_(H) (250 MHz; D₂O): 7.16 (2H, d, 9, ArH), 6.97 (2H, d, 9, ArH), 6.72(2H, s, ArH), 3.08 (12H, s, NCH₃).

Samples of the Medex™ starting material and the products of Synthesis 5(MTC-5° C.-crude) and Synthesis 6 (MTC-5° C.-recrystallised) werecompared using HPLC. Chromatograms for the MTC-5° C.-crude and MTC-5°C.-recrystallised are shown in FIG. 3 and FIG. 4, respectively. Theimpurity levels are summarised in the following Table. Azure A, Azure,B, Azure C, and MVB (Methylene Violet Bernthsen) are common undesirableimpurities typically found in samples of MTC.

TABLE 3 MTC Azure B Azure A Azure C MVB Other Sample w/w % w/w % w/w %w/w % w/w % w/w % Medex ™ 93.76 5.46 0.18 0.23 0.09 0.28 MTC-5° C. 99.350.31 0 0 0 0.34 (crude) MTC-5° C. 99.73 0.27 0 0 0 0 (recrystal- lised)

Samples of the Medex™ starting material and the products of Synthesis 5(MTC-5° C.-crude) and Synthesis 6 (MTC-5° C.-recrystallised) wereanalysed for metal content using ion-coupled plasma mass spectrometry(ICPMS). The metal contents are compared with the current EuropeanPharmacopoeia (EP) limits in the following Table.

TABLE 4 EP Limits (μg/g) MTC- (Version 5° C. MTC-5° C. Element EP5.4)Medex ™ (crude) (recrystallised) Aluminium (Al) 100 8.0 8.3 2.1 Cadmium(Cd) 1 <0.12 <0.04 <0.04 Chromium (Cr) 100 125 (*) 3.7 <0.76 Copper (Cu)300 269 1.6 <0.69 Tin (Sn) 10 <0.90  1.0 (*) 0.7 Iron (Fe) 200 92.2  687(*) 18.9 Manganese (Mn) 10 <0.17 4.9 0.3 Molybdenum (Mo) 10 <0.47 0.50.3 Nickel (Ni) 10 <0.65 <0.51 <0.51 Lead (Pb) 10 1.0 0.3 0.4 Zinc (Zn)100 <1.25 10.5 (*) 6.9 (*) denotes a failure to meet the EuropeanPharmacopoeia (EP) limits.

Synthesis 7 3,7-Dimethylamino-10H-phenothiazine

To a 250 cm³ round bottom flask placed under an atmosphere of argon wasadded methylthioninium chloride (Medex™) (MTC.3H₂O, MW 373.90, 10 g,26.7 mmol) and ethanol (100 cm³). Hydrazine hydrate (NH₂NH₂.H₂O, MW32.05, 3.0 cm³, 68.0 mmol) was added in portions. The mixture was heatedto 40° C. and stirred for 30 minutes. The resulting green suspension wascooled to 5° C. using an ice-water bath and filtered by canular underargon, washed once with ethanol (20 cm³), and dried under vacuum at roomtemperature to give the title compound as a light green/grey solid. Theproduct was used without further purification in Synthesis 8.

Synthesis 8 3,7-Dimethylamino-10-acetyl-phenothiazine

The 3,7-dimethylamino-10H-phenothiazine obtained in Synthesis 7 wasplaced in a 250 cm³ round bottom flask and acetic anhydride ((H₃CCO)₂O,40 cm³) and pyridine (10 cm³) were added, and the resulting solution washeated at 100° C. for 18 hours with stirring. The reaction mixture wascooled to room temperature and then poured carefully into ice-waterwhile stirring to give a light brown solid, which was filtered using astandard Buchner funnel apparatus with a water aspirator, washed oncewith water (100 cm³), and dried in an oven at 60° C. for 3 hours toyield the title compound, which was recrystallised from ethanol bydissolving it in hot ethanol (100 cm³), adding activated charcoal (1 g),filtering to remove the charcoal, cooling the solution to 5° C. using anice-water bath so that a precipitate formed (colourless crystals), andfiltering using a standard Buchner filter with a water aspirator inorder to collect the crystals. The crystals where then dried in an ovenat 60° C. for 3 hours (MW 327.45, 4.25 g, 13.0 mmol, yield 49%).

δ_(H) (250 MHz; CDCl₃) 2.16 (3H, s, CH₃), 2.92 (12H, s, NCH₃), 6.60-6.62(2H, d, ArH), 6.70-6.73 (2H, d, ArH), 7.08-7.47 (2H, brd s, ArH).

Synthesis 9 Methylthioninium Chloride (MTC)

3,7-Dimethylamino-10-acetyl-phenothiazine obtained in Synthesis 8 (MW327.45, 1 g, 3.05 mmol) was placed in a 50 cm³ round bottom flask, andwater (10 cm³) and hydrochloric acid (10 M, 3 cm³) were added, and theresulting solution was heated at 80° C. for 1 hour with stirring to givea green solution. The reaction mixture was cooled to 5° C. using anice-water bath, and divided into six fractions (6×2 cm³). Individualfractions (2 cm³) were used in each of the following synthesis methods.

Method A: To one fraction (2 cm³), aqueous iron chloride hexahydrate(FeCl₃.6H₂O, MW 270.30, 0.17 g, 0.63 mmol, 2 cm³) was added, giving animmediate deep blue colour. After 10 minutes, the mixture was filteredand the precipitate was washed with water (˜2 cm³) and air-dried to givethe title compound as a green powder (MW 319.86, 0.16 g, 0.51 mmol,100%).

Method B: To one fraction (2 cm³), Amberlite resin I.R. 120 (0.1 g) wasadded, giving an immediate blue colour. The mixture was heated to 60° C.for 30 minutes, and then hot-filtered to remove the resin. Hydrochloricacid (10 M, 4 drops) was added, and the mixture was allowed to stand for4 days. Then the mixture was filtered and the precipitate was washedwith water (˜2 cm³) and air-dried to give the title compound as greencrystals (MW 319.86, 0.14 g, 0.44 mmol, 86%).

The purity levels for the products are summarised in the followingTable. Note that the Medex™ starting material had a corresponding purityof 93.86%.

TABLE 5 Purity Method Reagent (wt %) A Iron chloride hexahydrate 99.09 BAmberlite Resin I.R. 120 98.94

Synthesis 10 3,7-Dimethylamino-10-acetyl-phenothiazine

To a 250 cm³ round bottom flask placed under an atmosphere of argon wasadded methylthioninium chloride (Medex™) (MTC.3H₂O, MW 373.90, 10 g,26.7 mmol) and acetonitrile (50 cm³). Hydrazine hydrate (NH₂NH₂.H₂O, MW50.06, 2.85 cm³, 58.7 mmol) was added in portions. The mixture washeated to 65° C. and stirred for 20 minutes. The resulting brownsuspension was cooled to 5° C. using an ice-water bath. Acetic anhydride((H₃CCO)₂O, MW 102.09, 25 cm³, 267 mmol) and N,N-diisopropylethylamine(MW 129.24, 9.3 cm³, 53.6 mmol) were added, and the resulting solutionwas heated at 100° C. for 2 hours with stirring. The reaction mixturewas cooled to 5° C. and had water (50 cm³) added while stirring to givea light green solid, which was filtered using a standard Buchner funnelapparatus with a water aspirator, washed with water (4×6 cm³), and driedin an oven at 60° C. for 3 hours to yield the title compound, which waspurified from hot ethanol (27 cm³), cooling the solution to 5° C. usingan ice-water bath so that a precipitate formed, and filtering using astandard Buchner filter with a water aspirator in order to collect thecrystals. The crystals where then dried in an oven at 60° C. for 3 hours(MW 327.45, 5.68 g, 17.4 mmol, yield 65%).

δ_(H) (250 MHz; CDCl₃) 2.16 (3H, s, CH₃), 2.92 (12H, s, NCH₃), 6.60-6.62(2H, d, ArH), 6.70-6.73 (2H, d, ArH), 7.08-7.47 (2H, brd s, ArH).

Synthesis 11 Methylthioninium Chloride (MTC)

3,7-Dimethylamino-10-acetyl-phenothiazine obtained in Synthesis 10 (MW327.45, 5 g, 15.27 mmol) was placed in a 50 cm³ round bottom flask, andwater (10 cm³) and hydrochloric acid (10 M, 5 cm³) were added, and theresulting solution was heated at 80° C. for 1 hour with stirring to givea green solution. The reaction mixture was cooled to 5° C. using anice-water bath, and divided into four even fractions (4×4 cm³). Onefraction (4 cm³) was used in each of the following synthesis methods.

Method A: To one fraction (4 cm³, 3.82 mmol), aqueous iron chloridehexahydrate (FeCl₃.6H₂O, MW 270.30, 2.06 g, 7.63 mmol, 15 cm³) solutionwas added, giving an immediate deep blue colour. After 30 minutes, themixture was filtered and the precipitate was washed with filtrate (˜5cm³) and air-dried to give the title compound as a green powder (MW319.86, 1.22 g, 3.82 mmol, 100%).

Method B: To one fraction (4 cm³, 3.82 mmol), isoamyl nitrite (MW 117.5,0.45 g, 3.82 mmol, in 8 cm³ of ethanol) was added, giving an immediatedeep blue colour. After 30 minutes, the mixture was filtered and theprecipitate was washed with filtrate (˜5 cm³) and air-dried to give thetitle compound as a green powder (MW 319.86, 0.64 g, 1.98 mmol, 52%).

Method C: To one fraction (4 cm³, 3.82 mmol), tert-butylamyl nitrite (MW103.1, 0.39 g, 3.82 mmol, in 8 cm³ of ethanol) was added, giving animmediate deep blue colour. After 30 minutes, the mixture was filteredand the precipitate was washed with filtrate (˜5 cm³) and air-dried togive the title compound as a green powder (MW 319.86, 0.67 g, 2.10 mmol,55%).

Method D: To one fraction (4 cm³, 3.82 mmol), Amberlite resin I.R. 120(1.25 g) was added, giving an immediate blue colour. The mixture washeated to 60° C. for 30 minutes, and then hot-filtered to remove theresin. Hydrochloric acid (10 M, 1 cm³) was added, and the mixture wasallowed to stand for 4 days. Then the mixture was filtered and theprecipitate was washed with filtrate (˜5 cm³) and air-dried to give thetitle compound as green crystals (MW 319.86, 1.05 g, 3.29 mmol, 86%).

The purity levels for the products are summarised in the followingTable. Note that the Medex™ starting material had a corresponding purityof 93.86%.

TABLE 5 Purity Method Reagent (wt %) A Iron chloride hexahydrate 99.09 BIsoamyl nitrite 98.89 C tert-Butyl nitrite 99.27 D Amberlite Resin I.R.120 98.94

Synthesis 12 3,7-Dimethylamino-10-acetyl-phenothiazine

To a 1000 cm³ round bottom flask placed under an atmosphere of argon wasadded methylthioninium chloride (Medex™) (MTC.3H₂O, MW 373.90, 50 g, 134mmol) and acetonitrile (250 cm³). Methylhydrazine (MeNHNH₂, MW 46.07,14.26 cm³, 268 mmol) was added in portions. The mixture was heated to35° C. and stirred for 20 minutes. The resulting brown suspension wascooled to 5° C. using an ice-water bath. Acetic anhydride ((H₃CCO)₂O, MW102.09, 101 cm³, 1.0 mol) and N,N-diisopropylethylamine (MW 129.24, 46.7cm³, 268 mmol) were added, and the resulting solution was heated at 100°C. for 2 hours with stirring. The reaction mixture was cooled to 5° C.and then had water (250 cm³) added while stirring to give a light greensolid, which was filtered using a standard Buchner funnel apparatus witha water aspirator, washed with water (4×30 cm³), and dried in an oven at60° C. for 3 hours to yield the title compound, which was purified fromhot ethanol (120 cm³) by cooling the solution to 5° C. using anice-water bath so that a precipitate formed and filtering using astandard Buchner filter with a water aspirator in order to collect thecrystals. The crystals were then dried in an oven at 60° C. for 3 hours(MW 327.45, 30.71 g, 93.8 mmol, yield 70%).

δ_(H) (250 MHz; CDCl₃) 2.16 (3H, s, CH₃), 2.92 (12H, s, NCH₃), 6.60-6.62(2H, d, ArH), 6.70-6.73 (2H, d, ArH), 7.08-7.47 (2H, brd s, ArH).

Synthesis 13 Methylthioninium Chloride (MTC)

3,7-Dimethylamino-10-acetyl-phenothiazine obtained in Synthesis 9 (MW327.45, 24.5 g, 75 mmol) was placed in a 500 cm³ round bottom flask, andwater (50 cm³) and hydrochloric acid (10 M, 25 cm³) were added, and theresulting solution was heated at 80° C. for 1 hour with stirring to givea green solution. The reaction mixture was cooled to 5° C. using anice-water bath. A cooled (5° C.) aqueous iron chloride hexahydratesolution (FeCl₃.6H₂O, MW 270.30, 40.6 g, 150 mmol, 300 cm³) was added,giving an immediate deep blue colour. After 30 minutes stirring, thesuspension was filtered and the precipitate was washed with filtrate(˜25 cm³) and air-dried for 30 minutes to give the crude title compoundas a purple solid. This material was dissolved in a mixture of water(480 cm³) and hydrochloric acid (10 M, 7.9 cm³) by heating to refluxbefore cooling to 25° C. The fine green needles were filtered, washedwith filtrate (50 cm³), and oven dried at 60° C. for 18 hours to givethe title compound containing 9% water (MW 319.86, 23.46 g, 74 mmol,98%). (The reported yield has been adjusted to represent anhydrous MTC).The mass of purified product that was obtained was 25.78 g, whichcontained 9% water. The characterisation data was the same as reportedabove for Synthesis 6.

Synthesis 14 3,7-Dinitrophenothiazine

Phenothiazine (MW 199.28, 20.00 g, 100 mmol), dichloromethane (100 cm³)and acetic acid (40 cm³) had sodium nitrite (MW 69.00, 20.7 g, 300 mmol)added and the mixture was stirred for 10 minutes at room temperature.Additional acetic acid (40 cm³), dichloromethane (100 cm³), and sodiumnitrite (MW 69.00, 20.7 g, 300 mmol) were then added. A further 120 cm³of acetic acid was added to try to break up the thick reaction mixture.The mixture was stirred for 2 hours. The suspension was filtered andwashed with 100 cm³ of each of ethanol, water, and finally ethanol togive a purple/brown solid. The residue was stirred in hot DMF (100 cm³)and allowed to cool before filtering to give the title dinitro product,which was washed with ethanol (150 cm³) and dried (MW 289.27, 24.88 g,86.0 mmol, 86%) to give a brown solid.

ν_(max) (KBr)/cm⁻¹ 3331 (NH), 3294 (NH), 3229 (NH), 3101 (CH), 3067(CH), 1602 (NO₂), 1558 (NO₂); δ_(H) (250 MHz; DMSO) 6.73-6.76 (2H, d, J9, ArH), 7.78 (2H, s, ArH), 7.89-7.85 (2H, d, J 9, ArH).

Synthesis 15 3,7-Dinitro-10-acetyl-phenothiazine

A solution of 3,7-dinitrophenothiazine (MW 289.27, 24.0 g, 82.96 mmol),acetic anhydride (MW 102.09, 151 g, 1.48 mol) and pyridine (100 cm³) wasstirred at reflux for 18 hours. The warm solution was then cooled toroom temperature and poured carefully over ice water. The precipitateformed was filtered to give the title compound (MW 331.26, 21.14 g,63.81 mmol, 77%) as a sand coloured solid which was recrystallised fromacetone to give light yellow needles.

ν_(max) (KBr)/cm⁻¹ 3091 (CH), 3063 (CH), 1680 (C═O), 1575 (NO₂), 1510(NO₂); δ_(H) (250 MHz; CDCl₃) 2.28 (3H, s, CH₃), 7.65-7.69 (2H, d, J 9,ArH), 8.22-8.26 (2H, dd, J 2.75, 8.75, ArH), 8.33-8.32 (2H, d, J 2.5,ArH); δ_(C) (62.9 MHz; CDCl₃) 168.2 (C═O), 146.3 (ArC), 143.3 (ArC),133.6 (ArC), 127.8 (ArC), 123.4 (ArC), 122.9 (ArC), 23.1 (CH₃); m/z (ES)331.0 (80%, [M]⁺).

Synthesis 16 3,7-diamino-10-acetyl-phenothiazine dihydrochloride

A mixture of 3,7-dinitro-10-acetyl-phenothiazine (MW 331.26, 2 g, 6.04mmol), palladium 10% on dry carbon (0.2 g) and tetrahydrofuran (20 cm³)was heated to 60° C. under an atmosphere of hydrogen and stirred at thistemperature for 18 hours. The mixture was cooled to room temperature,poured over celite filter aid, and washed with tetrahydrofuran (10 cm³).The THF filtrate was acidified with hydrochloric acid (10 M, 4 cm³) toprecipitate the product as a solid. The suspension was filtered to givethe title compound as a light green solid, which was dried at 60° C. for3 hours (MW 344.26, 4.3 mmol, 1.49 g, 72%).

δ_(H) (250 MHz; DMSO-d6) 2.12 (3H, s, CH₃), 7.30 (2H, d, J 8.25, ArH),7.45 (2H, s, ArH), 7.66 (2H, d, J 8.25, ArH); δ_(C) (62.9 MHz; CDCl₃)168.6 (C═O), 136.1 (ArC), 133.6 (ArC), 132.8 (ArC), 128.1 (ArC), 120.8(ArC), 22.6 (CH₃).

Synthesis 17 3,7-Dimethylamino-10-acetyl-phenothiazine

3,7-diamino-10-acetyl-phenothiazine dihydrochloride (MW 344.26, 2.59 g,7.55 mmol) was dissolved in water (7.5 cm³) in a conical flask (25 cm³)and to this solution was added sodium hydroxide solution (10%) to obtaina precipitate. The solid was filtered to give the free amine3,7-diamino-10-acetyl-phenothiazine (MW 271.34, 2.05 g, 7.55 mmol) whichwas dissolved in acetic acid (20 cm³) and p-formaldehyde (MW 30.03, 4.53g, 151 mmol) and sodium cyanoborohydride (MW 62.84, 4.74 g, 75.5 mmol)was added. The mixture was stirred at 50° C. for 2 hours, after whichwater (50 cm³) was added and the solid filtered to give crude product.This material was crystallized from ethanol (17 cm³) to give the titlecompound (MW 327.45, 0.75 g, 30%) as a colourless solid.

Mp 137° C.; ν_(max) (KBr)/cm⁻¹ 2910 (CH), 2876 (CH), 2856 (CH), 2799(CH), 1659 (C═O), 1596 (NO₂), 1502 (NO₂); δ_(H) (250 MHz; CDCl₃) 2.16(3H, s, CH₃), 2.93 (12H, s, NCH₃), 6.59-6.62 (2H, d, J 8.5, ArH),6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); δ_(C) (62.9MHz; CDCl₃) 170.3 (C═O), 148.9 (ArC), 127.2 (ArC), 127.1 (ArC), 127.0(ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH₃), 22.9 (CH₃); m/z (ES) 284.2(100%, [M−OAc]⁺), 328.1 (15%, [M+H]⁺), 350.1 (41%, [M+Na]⁺).

REFERENCES

A number of patents and publications are cited above in order to morefully describe and disclose the invention and the state of the art towhich the invention pertains. Full citations for these references areprovided below. Each of these references is incorporated herein byreference in its entirety into the present disclosure, to the sameextent as if each individual reference was specifically and individuallyindicated to be incorporated by reference.

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The invention claimed is:
 1. A diaminophenothiazinium compositioncomprising a diaminophenothiazinium compound of the following formula:

wherein: each of R¹ and R⁹ is independently —H, C₁₋₄alkyl, C₂₋₄alkenyl,or halogenated C₁₋₄alkyl; each of R^(3NA) and R^(3NB) is independentlyC₁₋₄alkyl, C₂₋₄alkenyl, or halogenated C₁₋₄alkyl; each of R^(7NA) andR^(7NB) is independently C₁₋₄alkyl, C₂₋₄alkenyl, or halogenatedC₁₋₄alkyl; and X is one or more anionic counter ions to achieveelectrical neutrality; wherein the composition is characterised by thefollowing by weight in the composition: at least 98% of thediaminophenothiazinium compound; less than 1% Azure B as impurity; lessthan 0.15% Azure A as impurity; less than 0.15% Azure C as impurity; andless than 0.05% Methylene Violet Bernthsen (MVB) as impurity.
 2. Adiaminophenothiazinium composition comprising a diaminophenothiaziniumcompound of the following formula:

wherein the composition is characterised by the following by weight inthe composition: at least 98% of the diaminophenothiazinium compound;less than 1% Azure B as impurity; less than 0.15% Azure A as impurity;less than 0.15% Azure C as impurity; and less than 0.05% MethyleneViolet Bernthsen (MVB) as impurity.
 3. The diaminophenothiaziniumcomposition of claim 2, having at least 99% of thediaminophenothiazinium compound by weight.
 4. The diaminophenothiaziniumcomposition of claim 2, having at least 99.5% of thediaminophenothiazinium compound by weight.
 5. The diaminophenothiaziniumcomposition of claim 2, having at least 99.6% of thediaminophenothiazinium compound by weight .
 6. Thediaminophenothiazinium composition of claim 2, having at least 99.7% ofthe diaminophenothiazinium compound by weight.
 7. Thediaminophenothiazinium composition of claim 2, having less than 0.5%Azure B as impurity by weight.
 8. The diaminophenothiazinium compositionof claim 2, having less than 0.1% Azure A as impurity by weight.
 9. Thediaminophenothiazinium composition of claim 2, having less than 0.05%Azure A as impurity by weight.
 10. The diaminophenothiaziniumcomposition of claim 2, having less than 0.1% Azure C as impurity byweight.
 11. The diaminophenothiazinium composition of claim 2, havingless than 0.05% Azure C as impurity by weight.
 12. Thediaminophenothiazinium composition of claim 2, having less than 0.02%MVB as impurity by weight.
 13. The diaminophenothiazinium composition ofclaim 2, having: less than 100 μg/g Aluminium (Al); less than 1 μg/gCadmium (Cd); less than 100 μg/g Chromium (Cr); less than 300 μg/gCopper (Cu); less than 10 μg/g Tin (Sn); less than 200 μg/g Iron (Fe);less than 10 μg/g Manganese (Mn); less than 1 μg/g Mercury (Hg); lessthan 10 μg/g Molybdenum (Mo); less than 10 μg/g Nickel (Ni); less than10 μg/g Lead (Pb); and less than 100 μg/g Zinc (Zn).
 14. Thediaminophenothiazinium composition of claim 2, having: less than 50 μg/gAluminium (Al); less than 0.5 μg/g Cadmium (Cd); less than 50 μg/gChromium (Cr); less than 150 μg/g Copper (Cu); less than 5 μg/g Tin(Sn); less than 100 μg/g Iron (Fe); less than 5 μg/g Manganese (Mn);less than 0.5 μg/g Mercury (Hg); less than 5 μg/g Molybdenum (Mo); lessthan 5 μg/g Nickel (Ni); less than 5 μg/g Lead (Pb); and less than 50μg/g Zinc (Zn).
 15. The diaminophenothiazinium composition of claim 1,wherein the diaminophenothiazinium compound is obtained by a method ofsynthesis comprising the steps of, in order: (a) purifying acorresponding acylated reagent compound the following formula, whereinR¹⁰ is independently saturated aliphatic C₁₋₅alkyl, phenyl,p-methoxyphenyl, or p-nitrophenyl:

(b)(i) deacylating the acylated reagent compound to give a correspondingdeacylated compound of the following formula:

(b)(ii) optionally purifying the deacylated compound; (c)(i) oxidizingthe deacylated compound to give the diaminophenothiazinium compound; and(c)(ii) optionally purifying the diaminophenothiazinium compound. 16.The diaminophenothiazinium composition of claim 2, obtained by a methodof synthesis comprising the steps of, in order: (a) purifying3,7-di(dimethylamino)-10-acetyl-phenothiazine; (b)(i) deacylating the3,7-di(dimethylamino)-10-acetyl-phenothiazine by reaction withhydrochloric acid to give 3,7-di(dimethylamino)-10H-phenothiazine;(b)(ii) optionally purifying the3,7-di(dimethylamino)-10H-phenothiazine; (c)(i) oxidizing the3,7-di(dimethylamino)-10H-phenothiazine by reaction with FeCl₃ to givemethylthioninium chloride; and (c)(ii) optionally purifying themethylthioninium chloride.
 17. The composition of claim 1, obtained by amethod of synthesis comprising the steps of, in order: (a) acylating acorresponding non-acylated precursor of a corresponding acylated reagentcompound to give the corresponding acylated reagent compound, whereinthe non-acylated precursor is a compound of the following formula:

and wherein the acylated reagent compound is a compound of the followingformula, wherein R¹⁰ is independently saturated aliphatic C₁₋₅alkyl,phenyl, p-methoxyphenyl, or p-nitrophenyl:

(b) purifying the acylated reagent compound; (c)(i) deacylating thepurified acylated reagent compound to give a corresponding deacylatedcompound of the following formula:

(c)(ii) optionally purifying the deacylated compound; (d)(i) oxidizingthe deacylated compound to give the diaminophenothiazinium compound; and(d)(ii) optionally purifying the diaminophenothiazinium compound. 18.The diaminophenothiazinium composition of claim 2, obtained by a methodof synthesis comprising the steps of, in order: (a) acylating3,7-di(dimethylamino)-10H-phenothiazine by reaction with (CH₃CO)₂O in abasic solvent to give 3,7-di(dimethylamino)-10-acetyl-phenothiazine; (b)purifying the 3,7-di(dimethylamino)-10-acetyl-phenothiazine; (c)(i)deacylating the 3,7-di(dimethylamino)-10-acetyl-phenothiazine byreaction with hydrochloric acid to give3,7-di(dimethylamino)-10H-phenothiazine; (c)(ii) optionally purifyingthe 3,7-di(dimethylamino)-10H-phenothiazine; (d)(i) oxidizing the3,7-di(dimethylamino)-10H-phenothiazine by reaction with FeCl₃ to givemethylthioninium chloride; and (d)(ii) optionally purifying themethylthioninium chloride.
 19. The diaminophenothiazinium composition ofclaim 1, obtained by a method of synthesis comprising the steps of, inorder: (a)(i) reducing a corresponding oxidized precursor of acorresponding non-acylated precursor of a corresponding acylated reagentcompound to give the non-acylated precursor of the acylated reagentcompound, wherein the oxidized precursor is a compound of the followingformula, wherein X^(a) is as defined for X, and may be the same as ordifferent than X:

and wherein the non-acylated precursor is a compound of the followingformula:

(a)(ii) optionally purifying the non-acylated precursor; (b) acylatingthe non-acylated precursor of the acylated reagent compound to give theacylated reagent compound, wherein the acylated reagent compound is acompound of the following formula, wherein R¹⁰ is independentlysaturated aliphatic C₁₋₅alkyl, phenyl, p-methoxyphenyl, orp-nitrophenyl:

(c) purifying the acylated reagent compound; (d)(i) deacylating theacylated reagent compound to give a corresponding deacylated compound ofthe following formula:

(d)(ii) optionally purifying the deacylated compound; (e)(i) oxidizingthe deacylated compound to give the diaminophenothiazinium compound; and(e)(ii) optionally purifying the diaminophenothiazinium compound. 20.The diaminophenothiazinium composition of claim 2, obtained by a methodof synthesis comprising the steps of, in order: (a)(i) reducingmethylthioninium chloride by reaction with one or more reducing reagentsselected from NaBH₄, MeNHNH₂, NH₂NH₂, and NH₂NH₂.H₂O, to give3,7-di(dimethylamino)-10H-phenothiazine; (a)(ii) optionally purifyingthe 3,7-di(dimethylamino)-10H-phenothiazine; (b) acylating the3,7-di(dimethylamino)-10H-phenothiazine by reaction with (CH₃CO)₂O in abasic solvent to give 3,7-di(dimethylamino)-10-acetyl-phenothiazine; (c)purifying the 3,7-di(dimethylamino)-10-acetyl-phenothiazine; (d)(i)deacylating the 3,7-di(dimethylamino)-10-acetyl-phenothiazine byreaction with hydrochloric acid to give3,7-di(dimethylamino)-10H-phenothiazine; (d)(ii) optionally purifyingthe 3,7-di(dimethylamino)-10H-phenothiazine; (e)(i) oxidizing the3,7-di(dimethylamino)-10H-phenothiazine by reaction with FeCl₃ to givemethylthioninium chloride; and (e)(ii) optionally purifying themethylthioninium chloride.
 21. The diaminophenothiazinium composition ofclaim 1, obtained by a method of synthesis comprising the steps of, inorder: (a) converting a corresponding acylated upstream precursor of acorresponding acylated reagent compound to the acylated reagentcompound, wherein the acylated upstream precursor is a compound of thefollowing formula, wherein R¹⁰ is independently saturated aliphaticC₁₋₅alkyl, phenyl, p-methoxyphenyl, or p-nitrophenyl:

and wherein the acylated reagent compound is a compound of the followingformula:

(b) purifying the acylated reagent compound; (c) deacylating theacylated reagent compound to give a corresponding deacylated compound ofthe following formula:

(d) optionally purifying the deacylated compound; (e)(i) oxidizing thedeacylated compound to give the diaminophenothiazinium compound; and(e)(ii) optionally purifying the diaminophenothiazinium compound. 22.The diaminophenothiazinium composition of claim 2, obtained by a methodof synthesis comprising the steps of, in order: (a) converting3,7-dinitro-10-acetyl-phenothiazine to3,7-di(dimethylamino)-10-acetyl-phenothiazine by (i) a nitro reductionstep comprising reaction with palladium and hydrogen and (ii) asubsequent amino alkylation step comprising reaction with sodium cyanoborohydride and paraformaldehyde; (b) purifying the3,7-di(dimethylamino)-10-acetyl-phenothiazine; (c)(i) deacylating the3,7-di(dimethylamino)-10-acetyl-phenothiazine by reaction withhydrochloric acid to give 3,7-di(dimethylamino)-10H-phenothiazine;(c)(ii) optionally purifying the3,7-di(dimethylamino)-10H-phenothiazine; (d)(i) oxidizing the3,7-di(dimethylamino)-10H-phenothiazine by reaction with FeCl₃ to givemethylthioninium chloride; and (d)(ii) optionally purifying themethylthioninium chloride.
 23. The diaminophenothiazinium composition ofclaim 1, obtained by a method of synthesis comprising the steps of, inorder: (a)(i) acylating a corresponding non-acylated upstream precursorof a corresponding acylated reagent compound to give the acylatedupstream precursor, wherein the non-acylated upstream precursor is acompound of the formula:

and wherein the acylated upstream precursor is a compound of thefollowing formula, wherein R¹⁰ is independently saturated aliphaticC₁₋₅alkyl, phenyl, p-methoxyphenyl, or p-nitrophenyl:

(a)(ii) optionally purifying the acylated upstream precursor; (b)converting the acylated upstream precursor to the acylated reagentcompound, wherein the acylated reagent compound is a compound of thefollowing formula:

(b) purifying the acylated reagent compound; (c)(i) deacylating theacylated reagent compound to give a corresponding deacylated compound ofthe following formula:

(c)(ii) optionally purifying the deacylated compound; (d)(i) oxidizingthe deacylated compound to give the diaminophenothiazinium compound; and(d)(ii) optionally purifying the diaminophenothiazinium compound. 24.The diaminophenothiazinium composition of claim 2, obtained by a methodof synthesis comprising the steps of, in order: (a)(i) acylating3,7-dinitro-10H-phenothiazine by reaction with (CH₃CO)₂O in a basicsolvent to give 3,7-dinitro-10-acetyl-phenothiazine; (a)(ii) optionallypurifying the 3,7-dinitro-10-acetyl-phenothiazine; (b) converting the3,7-dinitro-10-acetyl-phenothiazine to3,7-di(dimethylamino)-10-acetyl-phenothiazine by (i) a nitro reductionstep comprising reaction with palladium and hydrogen and (ii) asubsequent amino alkylation step comprising reaction with sodium cyanoborohydride and paraformaldehyde; (c) purifying the3,7-di(dimethylamino)-10-acetyl-phenothiazine; (d)(i) deacylating the3,7-di(dimethylamino)-10-acetyl-phenothiazine by reaction withhydrochloric acid to give 3,7-di(dimethylamino)-10H-phenothiazine;(d)(ii) optionally purifying the3,7-di(dimethylamino)-10H-phenothiazine; (e)(i) oxidizing the3,7-di(dimethylamino)-10H-phenothiazine by reaction with FeCl₃ to givemethylthioninium chloride; and (e)(ii) optionally purifying themethylthioninium chloride.
 25. A pharmaceutical composition comprisingthe diaminophenothiazinium composition of claim 1, and apharmaceutically acceptable carrier, diluent, or excipient.
 26. Apharmaceutical tablet or capsule comprising 20 to 300 mg of thediaminophenothiazinium composition of claim 1 and a pharmaceuticallyacceptable carrier, diluent, or excipient.
 27. A pharmaceutical tabletor capsule comprising 30 to 200 mg of the diaminophenothiaziniumcomposition of claim 2 and a pharmaceutically acceptable carrier,diluent, or excipient.
 28. A pharmaceutical composition comprising thediaminophenothiazinium composition of claim 2, and a pharmaceuticallyacceptable carrier, diluent, or excipient.